WO2021150076A1 - Pharmaceutical composition or health functional food for preventing or treating chronic obstructive pulmonary diseases - Google Patents
Pharmaceutical composition or health functional food for preventing or treating chronic obstructive pulmonary diseases Download PDFInfo
- Publication number
- WO2021150076A1 WO2021150076A1 PCT/KR2021/000939 KR2021000939W WO2021150076A1 WO 2021150076 A1 WO2021150076 A1 WO 2021150076A1 KR 2021000939 W KR2021000939 W KR 2021000939W WO 2021150076 A1 WO2021150076 A1 WO 2021150076A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- formula
- obstructive pulmonary
- chronic obstructive
- glycerol
- pharmaceutical composition
- Prior art date
Links
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 title claims abstract description 48
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 21
- 230000036541 health Effects 0.000 title claims abstract description 13
- 235000013376 functional food Nutrition 0.000 title claims abstract description 12
- 150000001875 compounds Chemical class 0.000 claims abstract description 92
- 239000004480 active ingredient Substances 0.000 claims abstract description 20
- 239000000126 substance Substances 0.000 claims abstract description 9
- 239000000203 mixture Substances 0.000 claims description 19
- 238000011282 treatment Methods 0.000 claims description 19
- 238000004519 manufacturing process Methods 0.000 claims description 18
- 230000002265 prevention Effects 0.000 claims description 9
- 210000004072 lung Anatomy 0.000 claims description 7
- 102000004127 Cytokines Human genes 0.000 claims description 5
- 108090000695 Cytokines Proteins 0.000 claims description 5
- 206010006458 Bronchitis chronic Diseases 0.000 claims description 3
- 206010014561 Emphysema Diseases 0.000 claims description 3
- 206010006451 bronchitis Diseases 0.000 claims description 3
- 208000007451 chronic bronchitis Diseases 0.000 claims description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 85
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 33
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 30
- 230000000052 comparative effect Effects 0.000 description 25
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 230000005764 inhibitory process Effects 0.000 description 20
- 210000003056 antler Anatomy 0.000 description 19
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 18
- 230000003110 anti-inflammatory effect Effects 0.000 description 18
- 239000002158 endotoxin Substances 0.000 description 17
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 17
- 229920006008 lipopolysaccharide Polymers 0.000 description 17
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 16
- 238000006243 chemical reaction Methods 0.000 description 15
- 238000002474 experimental method Methods 0.000 description 15
- 230000002401 inhibitory effect Effects 0.000 description 15
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 14
- 230000000694 effects Effects 0.000 description 14
- 239000000284 extract Substances 0.000 description 14
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 13
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 13
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 13
- JBYXPOFIGCOSSB-GOJKSUSPSA-N 9-cis,11-trans-octadecadienoic acid Chemical compound CCCCCC\C=C\C=C/CCCCCCCC(O)=O JBYXPOFIGCOSSB-GOJKSUSPSA-N 0.000 description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- GAKUNXBDVGLOFS-UHFFFAOYSA-N PLA Natural products CCCCCCCCCCCCCCCC(=O)OCC(COC(C)=O)OC(=O)CCCCCCCC=CCC=CCCCCC GAKUNXBDVGLOFS-UHFFFAOYSA-N 0.000 description 12
- 229940108924 conjugated linoleic acid Drugs 0.000 description 12
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 12
- 241000282994 Cervidae Species 0.000 description 11
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 11
- -1 beta-2 agonists Substances 0.000 description 11
- 239000003480 eluent Substances 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 11
- 230000002829 reductive effect Effects 0.000 description 11
- 239000000741 silica gel Substances 0.000 description 11
- 229910002027 silica gel Inorganic materials 0.000 description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 238000000605 extraction Methods 0.000 description 9
- 239000008213 purified water Substances 0.000 description 9
- 230000000391 smoking effect Effects 0.000 description 9
- 230000006433 tumor necrosis factor production Effects 0.000 description 9
- 101710085496 C-X-C motif chemokine 2 Proteins 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 102000004889 Interleukin-6 Human genes 0.000 description 8
- 108090001005 Interleukin-6 Proteins 0.000 description 8
- 102100038138 WD repeat-containing protein 26 Human genes 0.000 description 8
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 8
- 239000010410 layer Substances 0.000 description 8
- 210000000440 neutrophil Anatomy 0.000 description 8
- 210000003024 peritoneal macrophage Anatomy 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 7
- 239000012044 organic layer Substances 0.000 description 7
- 238000010898 silica gel chromatography Methods 0.000 description 7
- JJEYQTYWEVZYLY-UHFFFAOYSA-N (3-acetyloxy-2-hydroxypropyl) hexadecanoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(O)COC(C)=O JJEYQTYWEVZYLY-UHFFFAOYSA-N 0.000 description 6
- QHZLMUACJMDIAE-UHFFFAOYSA-N 1-monopalmitoylglycerol Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(O)CO QHZLMUACJMDIAE-UHFFFAOYSA-N 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 235000021314 Palmitic acid Nutrition 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 239000000411 inducer Substances 0.000 description 6
- 230000019189 interleukin-1 beta production Effects 0.000 description 6
- 230000017306 interleukin-6 production Effects 0.000 description 6
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- JVSFQJZRHXAUGT-UHFFFAOYSA-N 2,2-dimethylpropanoyl chloride Chemical compound CC(C)(C)C(Cl)=O JVSFQJZRHXAUGT-UHFFFAOYSA-N 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 5
- 239000012091 fetal bovine serum Substances 0.000 description 5
- 235000020778 linoleic acid Nutrition 0.000 description 5
- 238000004811 liquid chromatography Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- LMXZOKNDMCWYON-HZJYTTRNSA-N (12z,15z)-1,2,3-trihydroxyhenicosa-12,15-dien-4-one Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(=O)C(O)C(O)CO LMXZOKNDMCWYON-HZJYTTRNSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 4
- 241000208125 Nicotiana Species 0.000 description 4
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 235000019504 cigarettes Nutrition 0.000 description 4
- 239000000839 emulsion Substances 0.000 description 4
- 238000011156 evaluation Methods 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 241000411851 herbal medicine Species 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 231100000002 MTT assay Toxicity 0.000 description 3
- 238000000134 MTT assay Methods 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 150000008064 anhydrides Chemical class 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 230000013929 chemokine (C-X-C motif) ligand 2 production Effects 0.000 description 3
- 239000002026 chloroform extract Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000000469 ethanolic extract Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000005194 fractionation Methods 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 239000006187 pill Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000000779 smoke Substances 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000000638 solvent extraction Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- GAKUNXBDVGLOFS-DUZKARGPSA-N (1-acetyloxy-3-hexadecanoyloxypropan-2-yl) (9z,12z)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COC(C)=O)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC GAKUNXBDVGLOFS-DUZKARGPSA-N 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 241000283007 Cervus nippon Species 0.000 description 2
- 238000012286 ELISA Assay Methods 0.000 description 2
- 229920002683 Glycosaminoglycan Polymers 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- LJPZELFERXVJEK-NQLNTKRDSA-N [(12Z,15Z)-2,3-dihydroxy-4-oxohenicosa-12,15-dienyl] acetate Chemical compound C(CCCCCCC\C=C/C\C=C/CCCCC)(=O)C(O)C(O)COC(C)=O LJPZELFERXVJEK-NQLNTKRDSA-N 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
- WETWJCDKMRHUPV-UHFFFAOYSA-N acetyl chloride Chemical compound CC(Cl)=O WETWJCDKMRHUPV-UHFFFAOYSA-N 0.000 description 2
- 239000012346 acetyl chloride Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229940124630 bronchodilator Drugs 0.000 description 2
- 239000000168 bronchodilator agent Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000013932 chemokine (C-X-C motif) ligand 1 production Effects 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- 239000002274 desiccant Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 239000000428 dust Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 208000027866 inflammatory disease Diseases 0.000 description 2
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 2
- 125000002669 linoleoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- RNVYQYLELCKWAN-UHFFFAOYSA-N solketal Chemical compound CC1(C)OCC(CO)O1 RNVYQYLELCKWAN-UHFFFAOYSA-N 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- PFWLFWPASULGAN-UHFFFAOYSA-N 7-methylxanthine Chemical compound N1C(=O)NC(=O)C2=C1N=CN2C PFWLFWPASULGAN-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 241000282985 Cervus Species 0.000 description 1
- 241000974009 Cervus canadensis Species 0.000 description 1
- 241000283026 Cervus elaphus Species 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 206010017711 Gangrene Diseases 0.000 description 1
- 102100034221 Growth-regulated alpha protein Human genes 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 101001069921 Homo sapiens Growth-regulated alpha protein Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 241000234435 Lilium Species 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- MZNYWPRCVDMOJG-UHFFFAOYSA-N N-(1-naphthyl)ethylenediamine dihydrochloride Chemical compound [Cl-].[Cl-].C1=CC=C2C([NH2+]CC[NH3+])=CC=CC2=C1 MZNYWPRCVDMOJG-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- UTJMXUBBTOWNEK-DUZKARGPSA-N [(11Z,14Z)-2-(acetyloxymethyl)-2-hydroxy-3-oxoicosa-11,14-dienyl] hexadecanoate Chemical compound C(CCCCCCCCCCCCCCC)(=O)OCC(O)(COC(C)=O)C(CCCCCCC\C=C/C\C=C/CCCCC)=O UTJMXUBBTOWNEK-DUZKARGPSA-N 0.000 description 1
- BKFZUKXCZPTFOQ-DUZKARGPSA-N [(12Z,15Z)-1-acetyloxy-3-hydroxy-4-oxohenicosa-12,15-dien-2-yl] hexadecanoate Chemical compound C(CCCCCCC\C=C/C\C=C/CCCCC)(=O)C(O)C(OC(CCCCCCCCCCCCCCC)=O)COC(C)=O BKFZUKXCZPTFOQ-DUZKARGPSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000000809 air pollutant Substances 0.000 description 1
- 231100001243 air pollutant Toxicity 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940065524 anticholinergics inhalants for obstructive airway diseases Drugs 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229940124748 beta 2 agonist Drugs 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- RNFNDJAIBTYOQL-UHFFFAOYSA-N chloral hydrate Chemical compound OC(O)C(Cl)(Cl)Cl RNFNDJAIBTYOQL-UHFFFAOYSA-N 0.000 description 1
- 229960002327 chloral hydrate Drugs 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000000812 cholinergic antagonist Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000002934 diuretic Substances 0.000 description 1
- 230000001882 diuretic effect Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 150000002314 glycerols Chemical class 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 239000002035 hexane extract Substances 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000006058 immune tolerance Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 210000004283 incisor Anatomy 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 201000002364 leukopenia Diseases 0.000 description 1
- 231100001022 leukopenia Toxicity 0.000 description 1
- 238000011866 long-term treatment Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000074 matrix-assisted laser desorption--ionisation tandem time-of-flight detection Methods 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 125000001312 palmitoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000004526 pharmaceutical effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000002831 pharmacologic agent Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000011458 pharmacological treatment Methods 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000000737 potassium alginate Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001603 reducing effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- YEFOAORQXAOVJQ-RZFZLAGVSA-N schisandrol a Chemical compound C1[C@H](C)[C@@](C)(O)CC2=CC(OC)=C(OC)C(OC)=C2C2=C1C=C(OC)C(OC)=C2OC YEFOAORQXAOVJQ-RZFZLAGVSA-N 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 206010043554 thrombocytopenia Diseases 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
- A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
- A61K31/231—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having one or two double bonds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/314—Foods, ingredients or supplements having a functional effect on health having an effect on lung or respiratory system
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
- A23V2250/20—Natural extracts
- A23V2250/204—Animal extracts
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
- A23V2250/30—Other Organic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
Definitions
- the present invention relates to a pharmaceutical composition or health functional food for the prevention or treatment of chronic obstructive pulmonary disease.
- Chronic Obstructive Pulmonary Disease is a respiratory disease in which an abnormal inflammatory reaction occurs in the lungs due to inhalation of harmful particles or gases, leading to airflow limitation and difficulty in breathing.
- the most common cause of chronic obstructive pulmonary disease is smoking, and other air pollutants, fine dust, occupational dust, and inhalation of chemicals may be inhaled.
- the main symptoms of chronic obstructive pulmonary disease are dyspnea, cough, and phlegm, and chronic bronchitis, in which small airways are narrowed due to an inflammatory reaction in the lungs, and emphysema, in which lung tissue (alveoli) is destroyed.
- bronchodilators such as beta-2 agonists, anticholinergics, and methylxanthine, but bronchodilators prevent the progression of irreversible airflow restriction or cause inflammation of the small airways or lung parenchyma. It is known that it does not effectively inhibit Similar to asthma treatment, inhaled steroid preparations can be used to improve inflammation in chronic obstructive pulmonary disease, but many side effects may occur due to immunosuppression and immune tolerance, so it is suitable for chronic obstructive pulmonary disease patients who require long-term treatment don't
- Korean Patent Laid-Open No. 2010-0103999 discloses an herbal composition for preventing and treating chronic obstructive pulmonary disease, which contains a complex extract of Sukjihwang, Cheonmundong, Omija, Mokdanpi, Golden, Haengin, and Baekbu as active ingredients.
- Korean Patent Publication No. 2013-0107531 discloses a pharmaceutical composition for preventing and treating chronic obstructive pulmonary disease containing a lily extract as an active ingredient.
- Republic of Korea Patent Registration No. 1781310 discloses a complex formulation for improving chronic obstructive pulmonary disease, which contains a complex extract of guaruin, gold, hwangryeon and haengin as an active ingredient.
- Deer antlers (Cervi Parvum Cornu) are cut and dried deer antlers that are not ossified, such as Cervus nippon , Cervus elaphus , or Cervus canadensis , belonging to the deer and the genus Cervus.
- Deer antler has traditionally been widely used as a representative herbal medicine, and according to Donguibogam et al., it has a tonic action, a blood-keeping action, a gangrene action, an analgesic action, a hematopoietic action, a growth-promoting action, a heart failure treatment action, a functional enhancement action, a recovery from fatigue, and the body. It is known to have a wide variety of effects, such as enhancing vitality and strengthening the diuretic function of the kidneys.
- Deer antlers are known to contain various free amino acids, polysaccharides, glycosaminoglycans (GAGs), hyaluronic acid, keratin, sialic acid, cholesterol, fatty acids, phospholipids, and inorganic components.
- Korean Patent Laid-Open No. 1999-0044781 discloses that antler (maehwarok) is extracted with chloroform, and the chloroform extract is fractionated using silica gel column chromatography to isolate 5 types of monoacetyldiacylglycerol compounds, among which: Disclosed is the proliferation-promoting activity of 1-Palmitoyl-2-linoleoyl-3-acetyl glycerol (PLA, hereinafter, 'PLA glycerol') represented by the chemical formula.
- No. 2006-0047447 is an immunomodulatory agent and anticancer agent
- Patent Publication No. 2015-0021464 is a composition for inhibiting blood cancer or cancer metastasis
- 2015-0021465 is a composition for preventing or treating rheumatoid arthritis
- Patent Publication No. 2015- No. 0021472 discloses a composition for preventing or treating chronic obstructive pulmonary disease
- Patent Publication No. 2017-0005484 discloses a composition for treating leukopenia and thrombocytopenia.
- Korean Patent Laid-Open No. 2000-0059468 discloses that antler (maehwarok) is extracted with ethanol, and the ethanol extract is fractionated using silica gel column chromatography, and four types of phospholipids including the compound of the following formula are novel. The structure of the compound and its antifungal activity are disclosed.
- deer antler has various pharmacological activities, and its components are also very diverse, so continuous research on pharmacologically active ingredients that are not yet known is required.
- the present invention isolates a novel compound useful for the prevention and treatment of chronic obstructive pulmonary disease from deer antlers using solvent extraction and fractionation, and proposes a chemical synthesis method thereof to enable mass production of the novel compound, chronic obstructive pulmonary disease
- An object of the present invention is to provide a pharmaceutical composition and health functional food that can be used for the prevention and treatment of diseases.
- the present invention provides a pharmaceutical composition or health functional food for the prevention or treatment of chronic obstructive pulmonary disease, comprising at least one selected from the compounds represented by the following Chemical Formulas 1 to 4 as an active ingredient.
- the compound represented by Formula 1 and the compound represented by Formula 2 are isomers of each other, and the compound represented by Formula 3 and the compound represented by Formula 4 are also isomers of each other.
- the compounds of Formulas 1 to 4 may be isolated from antlers or chemically synthesized.
- the pharmaceutical composition or health functional food of the present invention may include, for example, a mixture of the compound represented by Formula 1 and the compound represented by Formula 2 above.
- the compound represented by Formula 1 and the compound represented by Formula 2 are isomers of each other.
- the pharmaceutical composition or health functional food of the present invention may include a mixture of a compound represented by Formula 3 and a compound represented by Formula 4 above.
- the compound represented by Formula 3 and the compound represented by Formula 4 are isomers of each other.
- the pharmaceutical composition or health functional food of the present invention may include a mixture of compounds represented by Formulas 1 to 4.
- the chronic obstructive pulmonary disease may be chronic bronchitis or emphysema, but is not limited thereto.
- the active ingredient may be to inhibit the production of cytokines in the lungs.
- the novel compounds of Examples 1 and 2 according to the present invention have no cytotoxicity, and the production of representative cytokines IL-6, IL-1 ⁇ , TNF- ⁇ , MIP2 and CXCL-1 is significantly higher than that of the control in animal experiments. It was confirmed that the cytokine production was significantly inhibited even when compared with the comparative example (PLA glycerol). Therefore, the novel compound according to the present invention can be usefully used as a composition for preventing, improving or treating chronic obstructive pulmonary disease.
- 1 is a diagram showing an extraction method using three organic solvents from antler according to the present invention.
- FIG. 2 is a diagram illustrating a method for fractionating an extract C2 extracted from deer antler according to the present invention.
- Figure 5 (A) is UV analysis data of fraction C2-2-Ea-Ms according to the present invention, (B) is UV analysis data of PLA glycerol of Comparative Example, (C) is conjugated linoleic acid (conjugated linoleic acid) of UV analysis data, and (D) is UV analysis data of linoleic acid.
- FIG. 6 is a graph showing the measurement results of cell viability (cell viability) according to the MTT assay of the novel compounds of Examples 1 and 2 of the present invention.
- FIG. 11 is a graph showing the neutrophil production inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention in COPD animal experiments.
- FIG. 13 is a graph showing the MIP2 inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention in a COPD-induced animal experiment.
- the present inventors In order to develop a pharmaceutical composition for the prevention or treatment of antler-derived inflammatory diseases, the present inventors analyzed and carried out the solvent extraction method and fractionation method of the prior literature from various angles to develop a novel compound having excellent physiological activity in inflammatory diseases by the following method was isolated, it was confirmed that the novel compound significantly inhibits the production of cytokines, which is a representative factor of chronic obstructive pulmonary disease, and a novel synthesis method of the compound was developed and the present invention was completed.
- the antler In order to extract the physiologically active ingredients from the antler, as shown in FIG. 1 , the antler (maehwarok) was sequentially extracted using three organic solvents: hexane, chloroform, and 70% ethanol, as shown in FIG. As described above, a novel compound having excellent anti-inflammatory activity was isolated by silica gel column chromatography and thin layer liquid chromatography.
- fraction C2-2-E 212 mg was taken. To 3.5 g of powdered silica gel, 10 ml of a mixed solution of n-Hx/EA/AcOH (20:1:0.5, v/v/v) was added and swollen, and the column was filled. 212 mg of fraction C2-2-E was dissolved in n-Hx/EA/AcOH (20:1:0.5, v/v/v) as an eluent and applied to a silica gel column.
- Figure 3 is the mass spectrometry data of the fraction C2-2-Ea-Ms
- Figure 4 is the LC data of the fraction C2-2-Ea-Ms
- Figure 5 (A) is the fraction C2-2-Ea according to the present invention -Ms UV analysis data
- (B) is UV analysis data of PLA glycerol of Comparative Example
- (C) is UV analysis data of conjugated linoleic acid
- (D) is UV analysis data of linoleic acid.
- the mass of the active ingredient of the fraction C2-2-Ea-Ms of the present invention was the same as the known PLA glycerol (Compound KJ-3 of Patent Publication No. 1999-0044781) isolated from antler, but UV analysis As a result, it showed a completely different UV analysis pattern from PLA glycerol.
- the compound of the fraction C2-2-Ea-Ms of the present invention is conjugated linoleic acid. linoleoyl) was confirmed to have a novel structure.
- the anti-inflammatory physiologically active ingredient according to the present invention is a novel compound represented by the following formulas 1 to 4 in which an acetyl group, palmitoyl, and conjugated linoleoyl are bonded to a glycerol structure. It was only confirmed in the invention.
- the compound of Formula 5 is a glycerol derivative protected with a 1,3-diol compound.
- the reaction may be carried out under basic conditions such as triethylamine, and may be carried out through an anhydride reaction by adding pivaloyl halide to maximize the reaction activity of palmitic acid.
- 1-palmitoyl glycerol is synthesized.
- the equivalent weight (eq.) of palmitic acid and the compound of Formula 5 is preferably 0.9:1.1 to 1.1:0.9, but is not limited thereto.
- the equivalent of 1-palmitoyl glycerol and acetyl halide in step (b) is preferably 1:1 to 1:1.6, but is not limited thereto.
- conjugated linoleic acid in step (c) may be carried out through an anhydride reaction by adding a pivaloyl halide.
- the equivalent (eq.) of 1-palmitoyl-3-acetyl glycerol and conjugated linoleic acid is preferably 0.9:1.1 to 1.1:0.9, but is not limited thereto.
- Example 2 1 -Conjugated- lineoyl -2- palmitoyl Preparation of -3-acetyl glycerol (CPA glycerol)
- PLA glycerol was synthesized in the same manner as in Example 1 using Linoleic acid (cis-9,cis-12) instead of conjugated Linoleic acid (cis-9,trans-11/trans-10,cis-12).
- Linoleic acid (cis-9,cis-12) 14.0 g and triethylamine 10.8 g were added to 180 ml of n-Hexane, cooled to 0° C., and 7.0 g of pivaloyl chloride was slowly added dropwise. After completion of the dropwise addition, after stirring at the same temperature for 1 hour, 18.0 g of 1-Palmitoyl-3-acetyl glycerol and 1.0 g of 4-dimethylaminopyridine were added and stirred at room temperature for 10 hours.
- HBSS was injected intraperitoneally into mice to extract macrophages, and after centrifugation at 3,000 rpm for 5 minutes, 100 units/mL of penicillin/streptomycin was added to DMEM medium supplemented with 10% fetal bovine serum (FBS). was isolated, and was used for the experiment after 24 hours of incubation in an incubator at 37° C., 5% CO 2
- the cultured peritoneal macrophages were aliquoted at 3 ⁇ 10 5 cells/well in a 96-well plate by 100 uL and cultured overnight. After removing the medium, the compounds of Example 1 (PCA glycerol) and Example 2 (CPA glycerol) were treated in peritoneal macrophages by concentration (10 ⁇ g/ml, 100 ⁇ g/ml and 200 ⁇ g/ml, respectively), and then , 37° C., 5% CO 2 Incubated for 24 hours in an incubator.
- MTT 5mg/mL
- DMSO reagent dispensed 600uL each, and then left at room temperature for 30 minutes. Then, the absorbance (OD) was measured at 540 nm with a microplate reader.
- Cell viability (cell viability) of the compounds of Examples 1 and 2 according to the MTT assay was measured and shown as a graph in Table 2 and FIG. 6 below. As shown in Table 2 and FIG. 6, it can be seen that there is no significant difference in cell viability even at the highest 200 ⁇ g/ml treatment. Therefore, it was confirmed that the novel compound according to the present invention is not cytotoxic.
- Example 1 ( ⁇ g/ml)
- Example 2 ( ⁇ g/ml) 10 100 200 10 100 200 cell Survival rate (%) 100 ⁇ 1.2 100 ⁇ 2.1 98.3 ⁇ 1.9 98.1 ⁇ 1.2 100 ⁇ 0.9 98.3 ⁇ 1.7 98.0 ⁇ 2.8
- mouse peritoneal macrophages were adjusted to 3 ⁇ 10 5 cells/mL, inoculated in a 96-well plate, and cultured for 24 hours in Example 1 (PCA glycerol), Example 2 (CPA glycerol) and Comparative Example ( PLA glycerol) was treated with each concentration (10 ⁇ g/ml, 100 ⁇ g/ml and 200 ⁇ g/ml, respectively), and LPS (1 ⁇ g/ml) was treated. The normal group was untreated, and the control group was treated with only LPS (1 ⁇ g/ml) in abdominal macrophages. After 12 hours of incubation, the supernatant was obtained by centrifugation.
- PBST phosphate buffered saline
- biotinylated anti-mouse IL-6, IL-1 ⁇ , and TNF- ⁇ detection antibody and streptavidin-HRP conjugate were washed and diluted with PBST It was aliquoted and reacted at room temperature for 1 hour. After that, it was washed again with PBST, and an OPD solution was added, followed by dark reaction at room temperature for 30 minutes. After terminating the reaction with 2 NH 2 SO 4 , the absorbance was measured at 450 nm using a microplate reader.
- the IL-6 production amount of the compounds of Examples 1 and 2 was measured and shown in Table 3 below, and is shown in the graph of FIG. 7 . As shown in Table 3 and Figure 7, it can be seen that the compounds prepared in Examples 1 and 2 significantly reduced IL-6 production compared to the control, and had a significant IL-6 production inhibitory effect even when compared to Comparative Examples there is.
- Example 2 Comparative Example ( ⁇ g/ml) 10 100 200 10 100 200 10 100 200 IL-6 amount of production (pg/ml) 150 ⁇ 18 834 ⁇ 32 321 ⁇ 22 225 ⁇ 23 180 ⁇ 25 325 ⁇ 13 234 ⁇ 20 179 ⁇ 17 364 ⁇ 16 251 ⁇ 24 203 ⁇ 25
- the IL-1 ⁇ production amount of the compounds of Examples 1 and 2 was measured and shown in Table 4 below, and is shown in the graph of FIG. 8 . As shown in Tables 4 and 8, the compounds prepared in Examples 1 and 2 significantly reduced the amount of IL-1 ⁇ production compared to the control, and it can be seen that they also had a significant IL-1 ⁇ production inhibitory effect when compared with the comparative example. there is.
- Example 2 ( ⁇ g/ml) Comparative Example ( ⁇ g/ml) 10 100 200 10 100 200 10 100 200 IL-1 ⁇ amount of production (pg/ml) 25 ⁇ 1.8 72 ⁇ 3.5 40 ⁇ 2.9 28 ⁇ 2.3 26 ⁇ 2.8 40 ⁇ 1.7 32 ⁇ 2.4 28 ⁇ 1.7 43 ⁇ 2.2 31 ⁇ 2.4 31 ⁇ 3.1
- the TNF- ⁇ production amount of the compounds of Examples 1 and 2 was measured and shown in Table 5 below, and is shown in the graph of FIG. 9 . As shown in Table 5 and Figure 9, the compounds prepared in Examples 1 and 2 significantly reduced the amount of TNF- ⁇ production compared to the control, and it can be seen that also has a significant TNF- ⁇ production inhibitory effect when compared with the comparative example. there is.
- the cultured peritoneal macrophages were suspended in DMEM containing 10% FBS, and then aliquoted at 5 ⁇ 10 5 cells/well in a 96-well plate and cultured at 37° C., 5% CO 2 in an incubator for 24 hours, and fresh DMEM medium. After exchanging with peritoneal macrophages, each of the compounds of Examples 1 and 2 and Comparative Example was treated with each concentration (10 ⁇ g/ml, 100 ⁇ g/ml and 200 ⁇ g/ml, respectively) in peritoneal macrophages and LPS (1 ⁇ g/ml) After treatment, incubated for 24 hours.
- the supernatant was separated and centrifuged at 3000 rpm for 5 minutes, and the separated supernatant was dispensed into a new microplate.
- the normal group was untreated, and the control group was treated with only LPS (1 ⁇ g/ml) in abdominal macrophages.
- the nitrogen monoxide (NO) production rates of the compounds of Examples 1 and 2 were measured and shown in Table 6 below, and shown in the graph of FIG. 10 . As shown in Tables 6 and 10, the compounds prepared in Examples 1 and 2 significantly reduced the NO production rate compared to the control, and it can be seen that they have a significant NO production inhibitory effect even when compared to the comparative example.
- Chronic obstructive pulmonary disease was induced by injecting Cigarette smoking solution (CSS) into the lungs of mice.
- CCS Cigarette smoking solution
- Tobacco smoke condensate was collected in a smoking room with a temperature of 22 ⁇ °C and a relative humidity of 60 ⁇ 5% according to ISO 3402 regulations, and the smoking method was a butt length of 3mm or more, a smoking volume of 35.0 ⁇ 0.3ml according to ISO 3308 regulations; It was carried out under the conditions of a smoking time of 2.00 ⁇ 0.02 seconds and a smoking cycle of 60 ⁇ 0.5 seconds.
- a Cambridge filter in which the cigarette smoke condensate is collected is placed in isopropanol and shaken, left at room temperature for more than 8 hours, extracted and filtered, and then completely concentrated using a vacuum filtration concentrator and nitrogen gas to extract a standard tobacco extract ( CSS) was prepared.
- COPD chronic obstructive pulmonary disease
- Each of the compound of Example 1, the compound of Example 2, and the compound of Comparative Example was dissolved in 60 mg/kg of 0.5% CMC (carboxmethylcellulose sodium) and orally administered 1 hour before inhalation of the inducer organ.
- the experimental group was (i) a normal group without any treatment (Normal), (ii) a control group treated with an inducer (LPS+CSS), (iii) the compound of Example 1 was orally administered 1 hour before the trigger material treatment
- the administration group was administered, (iv) the administration group in which the compound of Example 2 was orally administered 1 hour before the inducer treatment, and (v) the administration group in which the compound of Comparative Example was orally administered 1 hour before the inducer substance treatment.
- the obtained BALF was centrifuged at 4°C, 2,000 rpm, for 5 minutes to separate the precipitated blood cells, then treated with Diff-Quick Stain (Romanowsky stain) three times, and then washed twice with PBS, and then 9 slides per group was produced and counted through an optical microscope at 400 magnification, and is shown in Table 7 and FIG. 11 below.
- Example 2 comparative example Neutrophil count (/ ⁇ l) 11.1 ⁇ 1.6 231.2 ⁇ 14.5 73.4 ⁇ 21.6 82.0 ⁇ 18.9 122.7 ⁇ 27.4 Inhibition rate (%) - - 68% 65% 47%
- cytokines such as TNF- ⁇ , MIP2, CXCL-1, CXCL-8, IL-1 ⁇ , and IL-6 are increased in the BALF of chronic obstructive pulmonary disease (COPD) patients.
- COPD chronic obstructive pulmonary disease
- the amount of TNF- ⁇ , MIP2, and CXCL1 secretion in BALF obtained in Experimental Example 4 was measured using an ELISA assay (Millipore, USA) to evaluate the COPD inhibitory effect.
- Anti-mouse TNF- ⁇ , MIP2 and CXCL-1 capture antibodies were aliquoted into microplates. Then, it was washed with phosphate buffered saline (PBST) containing 0.05% Tween 20, blocked with 10% FBS, washed with PBST, and BAL cells were seeded in the wells and reacted for 2 hours at room temperature. After the reaction, biotinylated anti-mouse TNF- ⁇ , MIP2, and CXCL-1 detection antibodies and streptavidin-HRP conjugate (streptavidin-horseradish peroxydase conjugate) that were washed and diluted with PBST were dispensed. The reaction was carried out at room temperature for 1 hour.
- PBST phosphate buffered saline
- streptavidin-HRP conjugate streptavidin-HRP conjugate
- TNF- ⁇ , MIP2 and CXCL-1 production amounts of the compounds of Examples 1 and 2 were measured, respectively, and shown in Tables 8 to 10 below, and shown in the graphs of FIGS. 12 to 14 .
- Example 2 comparative example TNF- ⁇ amount of production (pg/ml) 7.1 ⁇ 1.1 151.6 ⁇ 10.6 42.3 ⁇ 8.5 39.8 ⁇ 6.7 58.4 ⁇ 7.1 Inhibition rate (%) - - 72% 74% 61%
- the amount of TNF- ⁇ production in the COPD-induced control group was 151.6 ⁇ 10.6 pg/ml, which was significantly increased compared to 7.1 ⁇ 1.1 pg/ml in the normal group, but the compound administered group of Example 1 is 42.3 ⁇ 8.5 pg/ml, 72% inhibition compared to the control group, and the compound administered group of Example 2 was 39.8 ⁇ 6.7 pg/ml, 74% inhibition, which was significant in TNF- ⁇ production compared to the comparative example (61% inhibition). It was confirmed that there is a reduction effect.
- Example 2 comparative example MIP2 amount of production (pg/ml) 62.1 ⁇ 7.1 301.4 ⁇ 16.7 91.2 ⁇ 11.5 95.3 ⁇ 5.6 111.4 ⁇ 8.9 Inhibition rate (%) - - 70% 68% 63%
- the amount of MIP2 production in the COPD-induced control group was 301.4 ⁇ 16.7 pg/ml, which was significantly increased compared to 62.1 ⁇ 7.1 pg/ml in the normal group, but the compound administered group of Example 1 was 91.2 70% inhibition rate compared to the control group at ⁇ 11.5 pg/ml, the compound administered group of Example 2 had a significant reduction effect in MIP2 production compared to the comparative example (63% inhibition rate) with a 68% inhibition rate at 95.3 ⁇ 5.6 pg/ml This was confirmed.
- Example 2 comparative example CXCL-1 amount of production (pg/ml) 64.2 ⁇ 8.1 250.4 ⁇ 18.7 77.3 ⁇ 12.0 84.9 ⁇ 8.6 89.1 ⁇ 11.3 Inhibition rate (%) - - 69% 66% 64%
- the amount of CXCL-1 produced in the control group induced by COPD was 250.4 ⁇ 18.7 pg/ml, which was significantly increased compared to 64.2 ⁇ 8.1 pg/ml in the normal group, but the group administered with the compound of Example 1 is 77.3 ⁇ 12.0 pg/ml, with a 69% inhibition rate compared to the control group, and the compound administered group of Example 2 was 84.9 ⁇ 8.6 pg/ml with a 66% inhibition rate, which was significant in the amount of CXCL-1 production compared to the comparative example (64% inhibition rate). It was confirmed that there is a reducing effect.
- the compounds of Formulas 1 to 4 may be included as an active ingredient in a pharmaceutical composition.
- the pharmaceutical composition is in the form of a conventional pharmaceutical composition such as tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-drying agents, etc. and may be in various oral or parenteral formulations.
- additives used in pharmaceutical formulations such as diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be included.
- composition of the present invention can be administered in a pharmaceutically effective amount.
- pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the subject's type and severity, age, sex, and disease. The type, activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined.
- the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple.
- the compounds of Formulas 1 to 4 may be included as an active ingredient of a health functional food.
- health functional food is a food used as a non-pharmaceutical, and there is no particular limitation on the type of food, for example, tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilization. It may be in the form of a conventional pharmaceutical composition such as an aqueous solution, a non-aqueous solvent, a suspension, an emulsion, and a freeze-drying agent, and may be a food additive.
- the present invention relates to a pharmaceutical composition or health functional food for the prevention or treatment of chronic obstructive pulmonary disease, comprising a novel compound isolated from deer antler as an active ingredient.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Emergency Medicine (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pulmonology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rheumatology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Immunology (AREA)
- Virology (AREA)
- Zoology (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The present invention provides a pharmaceutical composition or health functional food for preventing or treating chronic obstructive pulmonary diseases, comprising, as an active ingredient, at least one selected from among compounds represented by chemical formulas 1 to 4.
Description
본 발명은 만성폐쇄성 폐질환의 예방 또는 치료용 약학적 조성물 또는 건강기능식품에 관한 것이다.The present invention relates to a pharmaceutical composition or health functional food for the prevention or treatment of chronic obstructive pulmonary disease.
만성폐쇄성 폐질환(Chronic Obstructive Pulmonary Disease: 'COPD')이란 유해 입자나 가스 흡입으로 폐에 비정상적인 염증반응이 발생하여 기류제한(airflow limitation)이 진행됨으로써 호흡곤란을 유발하는 호흡기 질환이다. 만성폐쇄성 폐질환의 가장 흔한 원인은 흡연이며, 그 밖에 대기 오염물질, 미세먼지, 직업성 분진, 화학물질 등의 흡입일 수 있다. 만성폐쇄성 폐질환의 주요 증상은 호흡곤란, 기침, 가래 등이며, 폐에 염증반응이 발생해 소기도가 좁아지는 만성 기관지염과 폐조직(폐포)이 파괴되는 폐기종이 대표적이다.Chronic Obstructive Pulmonary Disease ('COPD') is a respiratory disease in which an abnormal inflammatory reaction occurs in the lungs due to inhalation of harmful particles or gases, leading to airflow limitation and difficulty in breathing. The most common cause of chronic obstructive pulmonary disease is smoking, and other air pollutants, fine dust, occupational dust, and inhalation of chemicals may be inhaled. The main symptoms of chronic obstructive pulmonary disease are dyspnea, cough, and phlegm, and chronic bronchitis, in which small airways are narrowed due to an inflammatory reaction in the lungs, and emphysema, in which lung tissue (alveoli) is destroyed.
전세계적으로 3억 2900만 명(전세계 인구의 5%)에 가까운 사람들이 만성폐쇄성 폐질환을 앓는 것으로 보고되고 있고, 세계보건기구(World Health Organization: WHO)는 2020년쯤에는 사망원인 3위, 장애원인 5위로 부상할 것으로 예상하고 있다.It is reported that close to 329 million people worldwide (5% of the global population) suffer from chronic obstructive pulmonary disease, and the World Health Organization (WHO) ranks as the third leading cause of death by 2020, disability It is expected to rise to the top 5 cause.
만성폐쇄성 폐질환의 약물치료는 베타-2 작용제, 항콜린제, 메틸잔틴 등의 기관지확장제를 중심으로 처방이 권고되고 있으나, 기관지확장제는 비가역적인 기류제한의 진행을 막거나 소기도 또는 폐실질의 염증을 효과적으로 억제하지는 못하는 것으로 알려져 있다. 천식치료와 유사하게 만성폐쇄성 폐질환의 염증개선을 위한 흡입성 스테로이드 제제를 사용할 수 있으나 면역억제 및 면역관용 등으로 인한 많은 부작용이 발생할 수 있기 때문에 장기간 치료를 필요로 하는 만성폐쇄성 폐질환 환자에게는 적합하지 않다.For pharmacological treatment of chronic obstructive pulmonary disease, prescription is recommended focusing on bronchodilators such as beta-2 agonists, anticholinergics, and methylxanthine, but bronchodilators prevent the progression of irreversible airflow restriction or cause inflammation of the small airways or lung parenchyma. It is known that it does not effectively inhibit Similar to asthma treatment, inhaled steroid preparations can be used to improve inflammation in chronic obstructive pulmonary disease, but many side effects may occur due to immunosuppression and immune tolerance, so it is suitable for chronic obstructive pulmonary disease patients who require long-term treatment don't
따라서 COPD 환자의 예후가 악화되는 것을 억제하면서 증상을 장기적으로 조절할 수 있는 효과적이고 안전한 제제 개발의 필요성이 지속적으로 요구되고 있다.Therefore, there is a continuous need for the development of effective and safe formulations that can control the symptoms of COPD patients for a long time while suppressing the deterioration of the prognosis.
한편, 이러한 만성폐쇄성 폐질환을 예방, 치료하기 위하여 한약재를 이용한 다양한 연구가 진행되었다. 대한민국 특허공개 제2010-0103999호는 숙지황, 천문동, 오미자, 목단피, 황금, 행인, 백부의 복합 추출물을 유효성분으로 하는 만성폐쇄성 폐질환 예방 및 치료용 한약조성물을 개시하고 있다. 대한민국 특허공개 제2013-0107531호는 백합 추출물을 유효성분으로 함유하는 만성폐쇄성 폐질환 예방 및 치료용 약학적 조성물을 개시하고 있다. 대한민국 특허등록 제1781310호는 과루인, 황금, 황련 및 행인의 복합 추출물을 유효성분으로 하는 만성폐쇄성 폐질환 개선용 복합 제제를 개시하고 있다.On the other hand, in order to prevent and treat such chronic obstructive pulmonary disease, various studies using herbal medicines have been conducted. Korean Patent Laid-Open No. 2010-0103999 discloses an herbal composition for preventing and treating chronic obstructive pulmonary disease, which contains a complex extract of Sukjihwang, Cheonmundong, Omija, Mokdanpi, Golden, Haengin, and Baekbu as active ingredients. Korean Patent Publication No. 2013-0107531 discloses a pharmaceutical composition for preventing and treating chronic obstructive pulmonary disease containing a lily extract as an active ingredient. Republic of Korea Patent Registration No. 1781310 discloses a complex formulation for improving chronic obstructive pulmonary disease, which contains a complex extract of guaruin, gold, hwangryeon and haengin as an active ingredient.
녹용(Cervi Parvum Cornu)은 사슴과 사슴속(Cervus)에 속한 매화록(Cervus nippon), 마록(Cervus elaphus) 또는 대록(Cervus canadensis) 등의 골질화되지 않은 사슴뿔을 잘라 말린 것이다.Deer antlers (Cervi Parvum Cornu) are cut and dried deer antlers that are not ossified, such as Cervus nippon , Cervus elaphus , or Cervus canadensis , belonging to the deer and the genus Cervus.
녹용은 전통적으로 보약을 대표하는 한약재로 널리 이용되고 있으며, 동의보감 등에 따르면 강장작용, 보혈작용, 강정작용, 진통작용, 조혈작용, 생장발육촉진작용, 심부전증 치료작용, 기능항진작용, 피로 회복, 신체 활력증강 및 신장의 이뇨기능 강화 등 매우 다양한 효능을 가지는 것으로 알려져 있다. 녹용에는 각종 유리 아미노산과 다당류, 글리코사미노글리칸(GAGs), 히알루론산, 케라틴, 시알산, 콜레스테롤, 지방산, 인지질, 무기질 성분 등이 함유되어 있는 것으로 알려져 있다.Deer antler has traditionally been widely used as a representative herbal medicine, and according to Donguibogam et al., it has a tonic action, a blood-keeping action, a gangrene action, an analgesic action, a hematopoietic action, a growth-promoting action, a heart failure treatment action, a functional enhancement action, a recovery from fatigue, and the body. It is known to have a wide variety of effects, such as enhancing vitality and strengthening the diuretic function of the kidneys. Deer antlers are known to contain various free amino acids, polysaccharides, glycosaminoglycans (GAGs), hyaluronic acid, keratin, sialic acid, cholesterol, fatty acids, phospholipids, and inorganic components.
녹용의 복용방법으로는, 녹용을 여러 종류의 생약재와 함께 열수 추출하여 그 여액을 복용하거나, 생약재들과 분쇄하여 가루로 만든 뒤 환(丸)으로 해서 복용하는 방법이 주로 이용되고 있으나, 용매추출 및 분획법을 이용하여 녹용에서 생리활성 유효성분을 추출하거나 분리하고자 하는 다양한 연구가 진행되었다.As a method of taking antler, hot water extraction of deer antler with various kinds of herbal medicines and taking the filtrate, or pulverizing with herbal medicines and taking them as pills are mainly used, but solvent extraction Various studies have been conducted to extract or isolate physiologically active ingredients from deer antlers using a fractionation method.
대한민국 특허공개 제1999-0044781호는 녹용(매화록)을 클로로포름으로 추출하고, 상기 클로로포름 추출물에 대하여 실리카겔 컬럼 크로마토그래피를 이용하여 분획하여 5종의 모노아세틸디아실글리세롤 화합물을 분리하고, 그 중 하기 화학식으로 표시되는 1-Palmitoyl-2-linoleoyl-3-acetyl glycerol(PLAG, 이하 'PLA glycerol')의 조혈모세포 및 혈소판 전구세포 증식 촉진활성을 개시하고 있으며, 이후 PLA glycerol 연구와 관련하여 대한민국 특허공개 제2006-0047447호는 면역조절제 및 항암제, 특허공개 제2015-0021464호는 혈액암 또는 암전이 억제용 조성물, 특허공개 제2015-0021465호는 류마티스 관절염의 예방 또는 치료용 조성물, 특허공개 제2015-0021472호는 만성폐쇄성 폐질환의 예방 또는 치료용 조성물, 특허공개 제2017-0005484호는 백혈구 감소증 및 혈소판 감소증 치료용 조성물을 개시하고 있다.Korean Patent Laid-Open No. 1999-0044781 discloses that antler (maehwarok) is extracted with chloroform, and the chloroform extract is fractionated using silica gel column chromatography to isolate 5 types of monoacetyldiacylglycerol compounds, among which: Disclosed is the proliferation-promoting activity of 1-Palmitoyl-2-linoleoyl-3-acetyl glycerol (PLA, hereinafter, 'PLA glycerol') represented by the chemical formula. No. 2006-0047447 is an immunomodulatory agent and anticancer agent, Patent Publication No. 2015-0021464 is a composition for inhibiting blood cancer or cancer metastasis, Patent Publication No. 2015-0021465 is a composition for preventing or treating rheumatoid arthritis, Patent Publication No. 2015- No. 0021472 discloses a composition for preventing or treating chronic obstructive pulmonary disease, and Patent Publication No. 2017-0005484 discloses a composition for treating leukopenia and thrombocytopenia.
한편, 대한민국 특허공개 제2000-0059468호는 녹용(매화록)을 에탄올로 추출하고, 상기 에탄올 추출물에 대하여 실리카겔 컬럼 크로마토그래피를 이용하여 분획하여 하기 화학식의 화합물을 비롯한 4종의 포스포리피드계 신규화합물의 구조 및 이의 항진균 활성에 대해 개시하고 있다.On the other hand, Korean Patent Laid-Open No. 2000-0059468 discloses that antler (maehwarok) is extracted with ethanol, and the ethanol extract is fractionated using silica gel column chromatography, and four types of phospholipids including the compound of the following formula are novel. The structure of the compound and its antifungal activity are disclosed.
녹용은 상술한 바와 같이 다양한 약리활성이 있고, 그 성분들도 매우 다양하여 아직까지 알려지지 않은 약리적 활성성분에 대한 지속적인 연구가 필요하다.As described above, deer antler has various pharmacological activities, and its components are also very diverse, so continuous research on pharmacologically active ingredients that are not yet known is required.
본 발명은 용매추출 및 분획법을 이용하여 녹용으로부터 만성폐쇄성 폐질환의 예방 및 치료에 유용한 신규 화합물을 분리하는 한편, 상기 신규 화합물의 대량 생산이 가능하도록 이의 화학적 합성방법을 제시함으로써, 만성폐쇄성 폐질환의 예방 및 치료에 이용될 수 있는 약학적 조성물 및 건강기능식품을 제공하는 데 그 목적이 있다.The present invention isolates a novel compound useful for the prevention and treatment of chronic obstructive pulmonary disease from deer antlers using solvent extraction and fractionation, and proposes a chemical synthesis method thereof to enable mass production of the novel compound, chronic obstructive pulmonary disease An object of the present invention is to provide a pharmaceutical composition and health functional food that can be used for the prevention and treatment of diseases.
본 발명은 하기 화학식 1 내지 4로 표시되는 화합물에서 선택되는 1종 이상을 유효성분으로 하는, 만성폐쇄성 폐질환의 예방 또는 치료용 약학적 조성물 또는 건강기능식품을 제공한다.The present invention provides a pharmaceutical composition or health functional food for the prevention or treatment of chronic obstructive pulmonary disease, comprising at least one selected from the compounds represented by the following Chemical Formulas 1 to 4 as an active ingredient.
[화학식 1][Formula 1]
[화학식 2][Formula 2]
[화학식 3][Formula 3]
[화학식 4][Formula 4]
상기 화학식 1로 표시되는 화합물 및 화학식 2로 표시되는 화합물은 서로 이성질체이고, 상기 화학식 3으로 표시되는 화합물 및 화학식 4로 표시되는 화합물 역시 서로 이성질체이다.The compound represented by Formula 1 and the compound represented by Formula 2 are isomers of each other, and the compound represented by Formula 3 and the compound represented by Formula 4 are also isomers of each other.
본 발명에 상기 화학식 1 내지 4의 화합물은 녹용으로부터 분리되거나 화학적으로 합성될 수 있다.In the present invention, the compounds of Formulas 1 to 4 may be isolated from antlers or chemically synthesized.
본 발명의 약학적 조성물 또는 건강기능식품은 예시적으로 상기 화학식 1로 표시되는 화합물 및 화학식 2로 표시되는 화합물의 혼합물을 포함할 수 있다. 상기 화학식 1로 표시되는 화합물과 화학식 2로 표시되는 화합물은 서로 이성질체이다.The pharmaceutical composition or health functional food of the present invention may include, for example, a mixture of the compound represented by Formula 1 and the compound represented by Formula 2 above. The compound represented by Formula 1 and the compound represented by Formula 2 are isomers of each other.
본 발명의 약학적 조성물 또는 건강기능식품은 다른 예시적으로 상기 화학식 3으로 표시되는 화합물 및 화학식 4로 표시되는 화합물의 혼합물을 포함할 수 있다. 상기 화학식 3으로 표시되는 화합물과 화학식 4로 표시되는 화합물은 서로 이성질체이다.As another example, the pharmaceutical composition or health functional food of the present invention may include a mixture of a compound represented by Formula 3 and a compound represented by Formula 4 above. The compound represented by Formula 3 and the compound represented by Formula 4 are isomers of each other.
본 발명의 약학적 조성물 또는 건강기능식품은 또 다른 예시적으로 상기 화학식 1 내지 4로 표시되는 화합물의 혼합물을 포함할 수 있다.As another example, the pharmaceutical composition or health functional food of the present invention may include a mixture of compounds represented by Formulas 1 to 4.
상기 만성폐쇄성 폐질환은 만성 기관지염 또는 폐기종인 것일 수 있으나, 이에 제한되지 않는다.The chronic obstructive pulmonary disease may be chronic bronchitis or emphysema, but is not limited thereto.
상기 유효성분은 폐에서 사이토카인의 생성을 억제하는 것일 수 있다.The active ingredient may be to inhibit the production of cytokines in the lungs.
본 발명에 따른 실시예 1 및 2의 신규 화합물은 세포독성이 없으며, 동물 실험에서 대조구에 비해 대표적 사이토카인들인 IL-6, IL-1β, TNF-α, MIP2 및 CXCL-1의 생성이 현저하게 억제되고, 비교예(PLA glycerol)와 대비할 때에도 사이토카인 생성이 유의적으로 억제되는 것이 확인되었다. 따라서 본 발명에 따른 신규 화합물은 만성폐쇄성 폐질환의 예방, 개선 또는 치료용 조성물로 유용하게 이용될 수 있다.The novel compounds of Examples 1 and 2 according to the present invention have no cytotoxicity, and the production of representative cytokines IL-6, IL-1β, TNF-α, MIP2 and CXCL-1 is significantly higher than that of the control in animal experiments. It was confirmed that the cytokine production was significantly inhibited even when compared with the comparative example (PLA glycerol). Therefore, the novel compound according to the present invention can be usefully used as a composition for preventing, improving or treating chronic obstructive pulmonary disease.
도 1은 본 발명에 따라 녹용으로부터 3가지 유기 용매를 이용한 추출 방법을 나타내는 다이어그램이다.1 is a diagram showing an extraction method using three organic solvents from antler according to the present invention.
도 2는 본 발명에 따라 녹용으로부터 추출한 추출물 C2의 분획 방법을 나타내는 다이어그램이다.2 is a diagram illustrating a method for fractionating an extract C2 extracted from deer antler according to the present invention.
도 3은 본 발명에 따른 분획물 C2-2-E-a-Ms의 질량분석 데이터이다.3 is mass spectrometry data of fraction C2-2-E-a-Ms according to the present invention.
도 4는 본 발명에 따른 분획물 C2-2-E-a-Ms의 LC 데이터이다.4 is LC data of fraction C2-2-E-a-Ms according to the present invention.
도 5의 (A)는 본 발명에 따른 분획물 C2-2-E-a-Ms의 UV분석 데이터이고, (B)는 비교예의 PLA glycerol의 UV분석 데이터이고, (C)는 공액 리놀레산(conjugated linoleic acid)의 UV분석 데이터이고, (D)는 리놀레산의 UV분석 데이터이다.Figure 5 (A) is UV analysis data of fraction C2-2-Ea-Ms according to the present invention, (B) is UV analysis data of PLA glycerol of Comparative Example, (C) is conjugated linoleic acid (conjugated linoleic acid) of UV analysis data, and (D) is UV analysis data of linoleic acid.
도 6은 본 발명의 실시예 1 및 실시예 2의 신규 화합물의 MTT assay에 따른 세포 생존율(cell viability) 측정 결과를 나타내는 그래프이다.6 is a graph showing the measurement results of cell viability (cell viability) according to the MTT assay of the novel compounds of Examples 1 and 2 of the present invention.
도 7은 본 발명의 실시예 1 및 실시예 2의 신규 화합물의 IL-6 생성 억제 효과를 나타내는 그래프이다. 7 is a graph showing the IL-6 production inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention.
도 8는 본 발명의 실시예 1 및 실시예 2의 신규 화합물의 IL-1β 생성 억제 효과를 나타내는 그래프이다. 8 is a graph showing the IL-1β production inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention.
도 9는 본 발명의 실시예 1 및 실시예 2의 신규 화합물의 TNF-α 억제 효과를 나타내는 그래프이다.9 is a graph showing the TNF-α inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention.
도 10은 본 발명의 실시예 1 및 실시예 2의 신규 화합물의 NO 형성 억제 효과를 나타내는 그래프이다.10 is a graph showing the NO formation inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention.
도 11은 COPD 동물실험에서 본 발명의 실시예 1 및 실시예 2의 신규 화합물호중구 생성 억제 효과를 나타내는 그래프이다.11 is a graph showing the neutrophil production inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention in COPD animal experiments.
도 12는 COPD 유발 동물실험에서 본 발명의 실시예 1 및 실시예 2의 신규 화합물의 TNF-α 억제 효과를 나타내는 그래프이다.12 is a graph showing the TNF-α inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention in COPD-induced animal experiments.
도 13은 COPD 유발 동물실험에서 본 발명의 실시예 1 및 실시예 2의 신규 화합물의 MIP2 억제 효과를 나타내는 그래프이다.13 is a graph showing the MIP2 inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention in a COPD-induced animal experiment.
도 14는 COPD 유발 동물실험에서 본 발명의 실시예 1 및 실시예 2의 신규 화합물의 CXCL-1 억제 효과를 나타내는 그래프이다.14 is a graph showing the CXCL-1 inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention in a COPD-induced animal experiment.
본 발명자는 녹용 유래 염증성 질환의 예방 또는 치료용 약학적 조성물을 개발하기 위하여, 선행문헌의 용매추출법 및 분획법을 다각도로 분석하고 실시하여 하기와 같은 방법으로 염증성 질환에 우수한 생리활성을 가지는 신규 화합물을 분리하고, 상기 신규 화합물이 만성폐쇄성 폐질환의 대표적 인자인 사이토카인의 생성을 유의적으로 억제하는 것을 확인하고, 상기 화합물의 신규 합성 방법을 개발하여 본 발명은 완성하였다.In order to develop a pharmaceutical composition for the prevention or treatment of antler-derived inflammatory diseases, the present inventors analyzed and carried out the solvent extraction method and fractionation method of the prior literature from various angles to develop a novel compound having excellent physiological activity in inflammatory diseases by the following method was isolated, it was confirmed that the novel compound significantly inhibits the production of cytokines, which is a representative factor of chronic obstructive pulmonary disease, and a novel synthesis method of the compound was developed and the present invention was completed.
녹용으로부터 생리활성 성분을 추출하기 위하여, 도 1에 도시된 바와 같이 녹용(매화록)을 헥산, 클로로포름, 70% 에탄올의 3가지 유기용매를 이용하여 순차적으로 추출을 실시하고, 도 2에 도시된 바와 같이 실리카겔 컬럼 크로마토그래피 및 박막 액체 크로마토그래피를 실시하여 항염증 활성이 우수한 신규 화합물을 분리하였다.In order to extract the physiologically active ingredients from the antler, as shown in FIG. 1 , the antler (maehwarok) was sequentially extracted using three organic solvents: hexane, chloroform, and 70% ethanol, as shown in FIG. As described above, a novel compound having excellent anti-inflammatory activity was isolated by silica gel column chromatography and thin layer liquid chromatography.
유기 용매를 통한 추출Extraction with organic solvents
시중에서 구입한 매화록(Cervus nippon) 2㎏을 분쇄하여 비이커에 넣고 1차 추출용매로서 헥산(n-Hexane) 9L를 넣은 다음, 80℃에서 2시간 가열하는 방식으로 2회 추출하고 여과하여 헥산 추출액을 얻은 다음, 상기 헥산 추출액을 감압 하에 증류, 건조시켜 추출물 C1 30.3g을 수득하였다.Pulverize 2 kg of commercially purchased Cervus nippon, put it in a beaker, add 9L of n-Hexane as the primary extraction solvent, and then extract twice by heating at 80°C for 2 hours, then filter and hexane After obtaining the extract, the hexane extract was distilled under reduced pressure and dried to obtain 30.3 g of extract C1.
헥산 추출 후 남은 잔사에 2차 추출용매로서 클로로포름(CHCl3) 9L를 넣은 다음, 80℃에서 2시간 동안 가열하여 2회 추출하고 여과하여 클로로포름 추출액을 얻은 다음, 클로로포름 추출액을 감압 하에 증류, 건조시켜 추출물 C2 17.9g을 수득하였다. After hexane extraction, 9 L of chloroform (CHCl 3 ) was added as a secondary extraction solvent to the residue remaining after extraction with hexane, and then extracted twice by heating at 80° C. for 2 hours, followed by filtration to obtain a chloroform extract, and then the chloroform extract was distilled and dried under reduced pressure. 17.9 g of extract C2 was obtained.
클로로포름 추출 후 남은 잔사에 3차 추출용매로서 70% 에탄올 9L를 넣은 다음, 80℃에서 5시간 동안 가열하여 2회 추출하고 여과하여 에탄올 추출액을 얻은 다음, 에탄올 추출액을 감압 하에 증류, 건조시켜 추출물 C3 89.2g을 수득하였다.After chloroform extraction, 9L of 70% ethanol as a tertiary extraction solvent was added to the residue remaining after extraction with chloroform, and then extracted twice by heating at 80° C. for 5 hours and filtered to obtain an ethanol extract. Then, the ethanol extract was distilled under reduced pressure and dried to extract C3 89.2 g were obtained.
상기에서 수득된 추출물 C1, C2 및 C3 각각에 대하여 항염증 활성 실험한 결과 추출물 C2에서 항염증 활성 효과가 높은 것으로 확인되어 추출물 C2을 대상으로 실리카겔 컬럼 크로마토그래피를 이용하여 생리활성 성분을 분획하였다.As a result of the anti-inflammatory activity experiment for each of the extracts C1, C2 and C3 obtained above, it was confirmed that the anti-inflammatory activity effect was high in the extract C2, and the bioactive component was fractionated using silica gel column chromatography for the extract C2.
실리카겔 컬럼 크로마토그래피Silica gel column chromatography
분말 실리카겔에 CHCl3/MeOH(500:1, v/v) 혼합용액을 가하여 팽윤시킨 후, 열린 컬럼에 충진시켰다. 상기 수득한 추출물 C2 17.9g을 CHCl3/MeOH(500:1, v/v)에 용해시켜 실리카겔이 충진된 컬럼에 적용시켰다. 컬럼에 용리액으로서 CHCl3/MeOH(500:2, v/v)을 흘러 넣어주면서 50㎖ 씩의 용출분획을 받은 다음, TLC(전개용매: CHCl3/MeOH=300:1)로 전개하여 요오드(I2)로 발색시킨 후 Rf치에 따라 분리하여 7개의 주요 분획을 분리하였다. 분리된 각각의 분획을 감압증류하여 건조시켜 분획물 C2-1 내지 C2-7을 수득하였다. 상기 7가지의 분획물에 대하여 항염증 활성 실험한 결과 분획물 C2-2에서 항염증 활성 효과가 높은 것으로 확인되어 분획물 C2-2를 대상으로 실리카겔 컬럼 크로마토그래피를 이용하여 생리활성 성분을 다시 분획하였다. A mixed solution of CHCl 3 /MeOH (500:1, v/v) was added to powder silica gel to swell, and then it was filled in an open column. 17.9 g of the obtained extract C2 was dissolved in CHCl 3 /MeOH (500:1, v/v) and applied to a column filled with silica gel. Elution fractions were received by 50 ml each while flowing CHCl 3 /MeOH (500:2, v/v) as an eluent into the column, and then developed with TLC (eluent: CHCl 3 /MeOH=300:1) to develop iodine ( After color development with I 2 ), 7 main fractions were separated by separation according to the Rf value. Each of the separated fractions was distilled under reduced pressure and dried to obtain fractions C2-1 to C2-7. As a result of the anti-inflammatory activity test for the seven fractions, it was confirmed that the anti-inflammatory activity effect was high in the fraction C2-2, and the bioactive components were re-fractionated using silica gel column chromatography for the fraction C2-2.
상기 분획물 C2-2 1.8g을 취하였다. 분말 실리카겔 20g에 헥산(n-Hx)/에틸아세테이트(EA)(50:1) 혼합용액 50㎖를 가하여 팽윤시켜 컬럼에 충진시켰다. 분획물 C2-2 1.8g을 용리액인 n-Hx/EA(50:1, v/v) 최소량에 용해시켜 실리카겔 컬럼에 적용시켰다. 상기 실리카겔 컬럼에 용리액을 흘려주면서 15㎖씩 용출분획을 받아 TLC(전개용매: n-Hx/EA=50:1, v/v)로 전개하고, 요오드로 발색시킨 후 Rf에 따라 분리하여 6개의 주요 분획을 분리하였다. 상기 분리된 각각의 분획을 감압증류시키고 건조시켜 분획물 C2-2-A 내지 C2-2-F을 수득하였다. 상기 6가지의 분획물에 대하여 항염증 활성 실험한 결과 분획물 C2-2-E에서 항염증 활성 효과가 높은 것으로 확인되어 분획물 C2-2-E를 대상으로 실리카겔 컬럼 크로마토그래피를 이용하여 생리활성 성분을 다시 분획하였다.1.8 g of the above fraction C2-2 was taken. To 20 g of powdered silica gel, 50 ml of a mixed solution of hexane (n-Hx)/ethyl acetate (EA) (50:1) was added, and the mixture was swollen and filled in a column. 1.8 g of Fraction C2-2 was dissolved in a minimum amount of n-Hx/EA (50:1, v/v) as an eluent and applied to a silica gel column. While flowing the eluent through the silica gel column, 15 ml of each eluted fraction was received, developed by TLC (eluent: n-Hx/EA=50:1, v/v), developed with iodine, and separated according to Rf, and separated according to Rf. The main fraction was isolated. Each of the separated fractions was distilled under reduced pressure and dried to obtain fractions C2-2-A to C2-2-F. As a result of the anti-inflammatory activity experiment for the six fractions, it was confirmed that the anti-inflammatory activity effect was high in the fraction C2-2-E. fractionated.
상기 분획물 C2-2-E 212㎎을 취하였다. 분말 실리카겔 3.5g에 n-Hx/EA/AcOH(20:1:0.5, v/v/v) 혼합용액 10㎖를 가하여 팽윤시켜 컬럼에 충진시켰다. 분획물 C2-2-E 212㎎을 용리액인 n-Hx/EA/AcOH(20:1:0.5, v/v/v)에 용해시켜 실리카겔 컬럼에 적용시켰다. 상기 실리카겔 컬럼에 용리액을 흘려주면서 10㎖씩 용출분획을 받아 TLC(전개용매: n-Hx/EA/AcOH=20:1:0.5, v/v/v)로 전개하고, 요오드로 발색시킨 후 Rf에 따라 분리하여 2개의 주요 분획을 분리하였다. 상기 분리된 각각의 분획을 감압증류시키고 건조시켜 각각 분획물 C2-2-E-a 및 C2-2-E-b을 수득하였다. 상기 2가지의 분획물에 대하여 항염증 활성 실험한 결과 분획물 C2-2-E-a에서 항염증 활성 효과가 높은 것으로 확인되어 분획물 C2-2-E-a를 대상으로 실리카겔 컬럼 크로마토그래피를 이용하여 생리활성 성분을 다시 분획하였다.212 mg of the fraction C2-2-E was taken. To 3.5 g of powdered silica gel, 10 ml of a mixed solution of n-Hx/EA/AcOH (20:1:0.5, v/v/v) was added and swollen, and the column was filled. 212 mg of fraction C2-2-E was dissolved in n-Hx/EA/AcOH (20:1:0.5, v/v/v) as an eluent and applied to a silica gel column. While flowing the eluent through the silica gel column, 10 ml of each eluted fraction was received and developed by TLC (eluent: n-Hx/EA/AcOH=20:1:0.5, v/v/v), developed with iodine, and then Rf was separated to separate two main fractions. Each of the separated fractions was distilled under reduced pressure and dried to obtain fractions C2-2-E-a and C2-2-E-b, respectively. As a result of the anti-inflammatory activity test for the two fractions, it was confirmed that the anti-inflammatory activity effect was high in the fraction C2-2-Ea, and the physiologically active ingredients were again recovered using silica gel column chromatography for the fraction C2-2-Ea. fractionated.
상기 분획물 C2-2-E-a 137㎎을 취하였다. 이 C2-2-E-a에 메탄올 5㎖을 가하여 혼합시켜 메탄올-가용성 분획물 C2-2-E-a-Ms)과 메탄올-불용성 분획물 C2-2-E-a-Mi)으로 분리하고 각각의 분획을 감압증류시키고 건조시켜 분획물 C2-2-E-a-Ms는 63㎎을 수득하였다. 상기 2가지의 분획물에 대하여 항염증 활성 실험한 결과 분획물 C2-2-E-a-Ms에서 항염증 활성 효과가 높은 것으로 확인되었다.137 mg of the fraction C2-2-E-a was taken. Methanol 5ml was added to this C2-2-Ea, mixed, and separated into a methanol-soluble fraction C2-2-Ea-Ms) and a methanol-insoluble fraction C2-2-Ea-Mi), and each fraction was distilled under reduced pressure and dried. and 63 mg of the fraction C2-2-Ea-Ms was obtained. As a result of an anti-inflammatory activity test for the two fractions, it was confirmed that the fraction C2-2-E-a-Ms had a high anti-inflammatory activity.
후술하는 실험예 2에 따른 방법을 통하여 상기 분획물의 항염증 억제 평가를 수행하여 그 결과를 하기 표 1에 나타내었다.Anti-inflammatory inhibition evaluation of the fraction was performed through the method according to Experimental Example 2 to be described later, and the results are shown in Table 1 below.
|
분획물fraction
(100㎍/ml)(100㎍/ml) |
IL-6 생성량IL-6 production
(pg/ml)(pg/ml) |
IL-1β 생성량IL-1β production
(pg/ml)(pg/ml) |
TNF-α 생성량TNF-α production
(pg/ml)(pg/ml) |
| C2-2 C2-2 | 453453 | 5959 | 283283 |
| C2-2-E C2-2-E | 402402 | 4343 | 233233 |
| C2-2-E-a C2-2-E-a | 321321 | 3636 | 197197 |
| C2-2-E-a-Ms C2-2-E-a-Ms | 253253 | 3434 | 162162 |
상기 표 1에 보이는 바와 같이, 분획물이 소분획될수록 염증성 사이토카인들인 IL-6, IL-1β, TNF-α의 생성이 억제됨을 알 수 있다. 분획물 중 가장 염증 억제 활성이 높은 분획물 C2-2-E-a-Ms의 유효성분을 확인하기 위하여 MALDI-TOF/TOF 질량분석기(Bruker UltraflextremeTM, German), 액체 크로마토그래피(LC) 및 UV 분석기를 이용하여 성분 분석을 실시하였다.As shown in Table 1, it can be seen that the smaller the fraction, the more inhibited the production of inflammatory cytokines IL-6, IL-1β, TNF-α. In order to confirm the active ingredient of the fraction C2-2-Ea-Ms, which has the highest anti-inflammatory activity among the fractions, MALDI-TOF/TOF mass spectrometer (Bruker Ultraflextreme TM , German), liquid chromatography (LC) and UV analyzer were used. Component analysis was performed.
도 3은 분획물 C2-2-E-a-Ms의 질량분석 데이터이고, 도 4는 분획물 C2-2-E-a-Ms의 LC 데이터이고, 도 5의 (A)는 본 발명에 따른 분획물 C2-2-E-a-Ms의 UV분석 데이터, (B)는 비교예의 PLA glycerol의 UV분석 데이터, (C)는 공액 리놀레산(conjugated linoleic acid)의 UV분석 데이터, (D)는 리놀레산의 UV분석 데이터이다.Figure 3 is the mass spectrometry data of the fraction C2-2-Ea-Ms, Figure 4 is the LC data of the fraction C2-2-Ea-Ms, Figure 5 (A) is the fraction C2-2-Ea according to the present invention -Ms UV analysis data, (B) is UV analysis data of PLA glycerol of Comparative Example, (C) is UV analysis data of conjugated linoleic acid, (D) is UV analysis data of linoleic acid.
본 발명의 분획물 C2-2-E-a-Ms의 유효성분의 질량을 분석한 결과, 질량은 녹용으로부터 분리된 공지의 PLA glycerol(특허공개 제1999-0044781호의 화합물 KJ-3)과 동일하였으나, UV분석 결과 PLA glycerol과는 전혀 다른 UV분석 패턴을 보였다. 도 5의 (C) 공액 리놀레산(Conjugated linoleic acid) UV 패턴, (D) 리놀레산(Linoleic acid)의 UV 패턴과 비교함으로써 본 발명의 분획물 C2-2-E-a-Ms의 화합물은 공액 리놀레오일(Conjugated linoleoyl)의 신규한 구조를 가지는 것이 확인되었다.As a result of analyzing the mass of the active ingredient of the fraction C2-2-Ea-Ms of the present invention, the mass was the same as the known PLA glycerol (Compound KJ-3 of Patent Publication No. 1999-0044781) isolated from antler, but UV analysis As a result, it showed a completely different UV analysis pattern from PLA glycerol. By comparing the UV pattern of (C) conjugated linoleic acid and (D) UV pattern of linoleic acid in FIG. 5, the compound of the fraction C2-2-Ea-Ms of the present invention is conjugated linoleic acid. linoleoyl) was confirmed to have a novel structure.
본 발명에 따른 상기 항염증 생리활성 유효성분은 글리세롤 구조에 아세틸기(acetyl), 팔미토일(palmitoyl), 공액 리놀레오일(conjugated linoleoyl)이 결합된 하기 화학식 1 내지 4로 표시되는 신규 화합물임이 본 발명에서 비로소 확인되었다.The anti-inflammatory physiologically active ingredient according to the present invention is a novel compound represented by the following formulas 1 to 4 in which an acetyl group, palmitoyl, and conjugated linoleoyl are bonded to a glycerol structure. It was only confirmed in the invention.
[화학식 1][Formula 1]
[화학식 2][Formula 2]
[화학식 3][Formula 3]
[화학식 4][Formula 4]
상기 화학식 1 및 화학식 2로 표시되는 PCA glycerol의 화학적 합성Chemical synthesis of PCA glycerol represented by Formula 1 and Formula 2
본 발명에 따른 화학식 1, 화학식 2의 화합물(PCA glycerol)은 하기 반응식 1에 따라 고수율로 제조될 수 있다.The compounds of Chemical Formulas 1 and 2 (PCA glycerol) according to the present invention can be prepared in high yield according to Scheme 1 below.
[반응식 1][Scheme 1]
본 발명에 따른 PCA glycerol은,PCA glycerol according to the present invention,
(a) 팔미트산과 상기 화학식 5로 표시되는 화합물을 염기 조건 하에서 반응시켜 1-팔미토일 글리세롤을 합성하는 단계;(a) reacting palmitic acid with the compound represented by Formula 5 under basic conditions to synthesize 1-palmitoyl glycerol;
(b) 상기 1-팔미토일 글리세롤과 아세틸 할라이드를 염기 조건 하에서 반응시켜 1-팔미토일-3-아세틸 글리세롤을 합성하는 단계; 및(b) reacting the 1-palmitoyl glycerol and acetyl halide under basic conditions to synthesize 1-palmitoyl-3-acetyl glycerol; and
(c) 상기 1-팔미토일-3-아세틸 글리세롤과 공액 리놀레산(Conjugated linoleic acid)를 염기 조건 하에서 반응시켜 1-팔미토일-2-공액 리놀레오일-3-아세틸 글리세롤(PCA glycerol)을 합성하는 단계를 포함한다.(c) reacting the 1-palmitoyl-3-acetyl glycerol with conjugated linoleic acid under basic conditions to synthesize 1-palmitoyl-2-conjugated linoleoyl-3-acetyl glycerol (PCA glycerol) includes steps.
상기 단계 (a)에서 화학식 5의 화합물은 1,3-디올 화합물로 보호된 글리세롤 유도체이다. 상기 반응은 트리에틸아민(triethylamine)와 같은 염기 조건 하에서 실시될 수 있고, 팔미트산의 반응활성을 극대화시키기 위하여 피바로일 할라이드(Pivaloyl halide)를 첨가하여 anhydride reaction을 통해 실시될 수 있다. 반응 후 염산 등을 사용하여 탈보호시키면 1-팔미토일 글리세롤이 합성된다. 팔미트산과 화학식 5의 화합물의 당량(eq.)은 0.9:1.1 ~ 1.1:0.9가 바람직하나 이에 제한되는 것은 아니다.In step (a), the compound of Formula 5 is a glycerol derivative protected with a 1,3-diol compound. The reaction may be carried out under basic conditions such as triethylamine, and may be carried out through an anhydride reaction by adding pivaloyl halide to maximize the reaction activity of palmitic acid. After the reaction, when deprotected using hydrochloric acid, 1-palmitoyl glycerol is synthesized. The equivalent weight (eq.) of palmitic acid and the compound of Formula 5 is preferably 0.9:1.1 to 1.1:0.9, but is not limited thereto.
상기 단계 (b)에서 1-팔미토일 글리세롤과 아세틸 할라이드의 당량은 1:1 ~ 1:1.6가 바람직하나 이에 제한되는 것은 아니다.The equivalent of 1-palmitoyl glycerol and acetyl halide in step (b) is preferably 1:1 to 1:1.6, but is not limited thereto.
상기 단계 (c)에서 공액 리놀레산의 반응활성을 극대화시키기 위하여 피바로일 할라이드(Pivaloyl halide)를 첨가하여 anhydride reaction을 통해 실시될 수 있다. 1-팔미토일-3-아세틸 글리세롤과 공액 리놀레산의 당량(eq.)은 0.9:1.1 ~ 1.1:0.9가 바람직하나 이에 제한되는 것은 아니다.In order to maximize the reaction activity of the conjugated linoleic acid in step (c), it may be carried out through an anhydride reaction by adding a pivaloyl halide. The equivalent (eq.) of 1-palmitoyl-3-acetyl glycerol and conjugated linoleic acid is preferably 0.9:1.1 to 1.1:0.9, but is not limited thereto.
한편, 본 발명에 따른 화학식 3, 화학식 4의 화합물(CPA glycerol)은 하기 반응식 2에 따라 고수율로 제조될 수 있다.Meanwhile, the compounds of Chemical Formulas 3 and 4 (CPA glycerol) according to the present invention can be prepared in high yield according to the following Reaction Scheme 2.
[반응식 2][Scheme 2]
본 발명에 따른 CPA glycerol은,CPA glycerol according to the present invention,
(a) 공액 리놀레산(Conjugated linoleic acid)과 상기 화학식 5로 표시되는 화합물을 염기 조건 하에서 반응시켜 1-공액 리놀레오일-글리세롤을 합성하는 단계;(a) reacting conjugated linoleic acid with the compound represented by Formula 5 under basic conditions to synthesize 1-conjugated linoleoyl-glycerol;
(b) 상기 1-공액 리놀레오일-글리세롤과 아세틸 할라이드를 염기 조건 하에서 반응시켜 1-공액 리놀레오일-3-아세틸 글리세롤을 합성하는 단계; 및(b) reacting the 1-conjugated linoleoyl-glycerol with an acetyl halide under basic conditions to synthesize 1-conjugated linoleoyl-3-acetyl glycerol; and
(c) 상기 1-공액 리놀레오일-3-아세틸 글리세롤과 팔미트산을 염기 조건 하에서 반응시켜 1-공액 리놀레오일-2-팔미토일-3-아세틸 글리세롤(CPA glycerol)을 합성하는 단계를 포함한다. 상기 단계 (a), (c)에서 anhydride reaction을 위해 피바로일 할라이드(Pivaloyl halide)를 첨가할 수 있다.(c) reacting the 1-conjugated linoleoyl-3-acetyl glycerol with palmitic acid under basic conditions to synthesize 1-conjugated linoleoyl-2-palmitoyl-3-acetyl glycerol (CPA glycerol); include Pivaloyl halide may be added for the anhydride reaction in steps (a) and (c).
이하, 실시예를 통하여 본 발명의 신규 화합물의 합성 방법을 구체적으로 설명한다.Hereinafter, a method for synthesizing the novel compound of the present invention will be described in detail through Examples.
실시예 Example
1: 11:1
--
PalmitoylPalmitoyl
-2-conjugated--2-conjugated-
linoleoyllinoleoyl
-3-acetyl glycerol (PCA glycerol)의 제조Preparation of -3-acetyl glycerol (PCA glycerol)
(1) 1-Palmitoyl glycerol의 제조(1) Preparation of 1-Palmitoyl glycerol
Methylene chloride(MC) 200ml에 Palmitic acid 20.0g과 triethylamine 17.0g을 첨가하고 0℃로 냉각한 후, Pivaloyl chloride 10.0g을 천천히 적가하고 2시간 동안 교반한 다음, Solketal 10.8g과 4-Dimethylaminopyridine 0.1g을 첨가한 후 실온에서 12시간 교반하고 정제수 200ml를 첨가하고 층분리 후 유기층은 진공 농축하였다. 농축 모액에 Methanol 200ml, 염산 20ml를 투입하고 실온에서 10시간 교반 후 n-hexane 300ml, 정제수 300ml를 투입하고 3시간 교반 후 여과하고 진공 건조하여 목적 화합물 21.9g (수율:85.0%)을 수득하였다.After adding 20.0 g of palmitic acid and 17.0 g of triethylamine to 200 ml of methylene chloride (MC), and cooling to 0°C, 10.0 g of pivaloyl chloride was slowly added dropwise, stirred for 2 hours, and then 10.8 g of Solketal and 0.1 g of 4-dimethylaminopyridine were added. After the addition, the mixture was stirred at room temperature for 12 hours, purified water 200ml was added, and after layer separation, the organic layer was concentrated in vacuo. 200ml of methanol and 20ml of hydrochloric acid were added to the concentrated mother liquor, and after stirring at room temperature for 10 hours, 300ml of n-hexane and 300ml of purified water were added, stirred for 3 hours, filtered, and vacuum dried to obtain 21.9g of the target compound (yield: 85.0%).
(2) 1-Palmitoyl-3-acetyl glycerol의 제조(2) Preparation of 1-Palmitoyl-3-acetyl glycerol
MC 200ml에 상기 수득된 1-Palmitoyl glycerol 20.0g과 Pyridine 33.5g, 4-Dimethylaminopyridine 0.2g을 첨가하고 1시간 동안 교반한 다음, Acetyl chloride 7.1g을 적가하고 5시간 교반하였다. 정제수 200ml를 투입하고 묽은 염산으로 중화하고 층분리 한 후 유기층을 MgSO4로 탈수한 후 진공 농축하고 n-hexane 100ml를 투입하고 10℃ 이하에서 결정화하여 여과 후 진공 건조하여 목적 화합물 18.5g (수율: 82.0%)을 수득하였다.20.0 g of 1-Palmitoyl glycerol obtained above, 33.5 g of pyridine, and 0.2 g of 4-dimethylaminopyridine were added to 200 ml of MC and stirred for 1 hour, then 7.1 g of Acetyl chloride was added dropwise and stirred for 5 hours. After adding 200 ml of purified water, neutralizing with dilute hydrochloric acid, separating the layers , dehydrating the organic layer with MgSO 4 , vacuum concentration, adding 100 ml of n-hexane, crystallization at 10° C. or lower, filtration, and vacuum drying the target compound 18.5 g (yield: 82.0%) was obtained.
(3) 1-Palmitoyl-2-C-linoleoyl-3-acetyl glycerol (PCA glycerol)의 제조(3) Preparation of 1-Palmitoyl-2-C-linoleoyl-3-acetyl glycerol (PCA glycerol)
n-Hexane 180ml에 conjugated Linoleic acid(cis-9,trans-11/trans-10,cis-12) 14.0g과 triethylamine 10.8g을 첨가하고 0℃로 냉각하고 Pivaloyl chloride 7.0g을 천천히 적가하였다. 적가 완료 후 동온도에서 1시간 교반 후, 1-Palmitoyl-3-Acetyl glycerol 18.0g과 4-Dimethylaminopyridine 1.0g을 첨가하고 실온에서 10시간 교반하였다. 정제수 180ml를 투입하고 층분리 하고 유기층은 MgSO4로 탈수한 후 진공 농축하고 실리카겔 컬럼 정제(용출액: n-Hx:EA=20:1, v/v)하여 목적화합물 26.1g (수율: 85%)을 수득하였다.14.0 g of conjugated linoleic acid (cis-9,trans-11/trans-10,cis-12) and 10.8 g of triethylamine were added to 180 ml of n-Hexane, cooled to 0° C., and 7.0 g of Pivaloyl chloride was slowly added dropwise. After completion of the dropwise addition, after stirring for 1 hour at the same temperature, 18.0 g of 1-Palmitoyl-3-Acetyl glycerol and 1.0 g of 4-Dimethylaminopyridine were added and stirred at room temperature for 10 hours. 180ml of purified water was added, the layers were separated, the organic layer was dehydrated with MgSO 4 , concentrated in vacuo, and purified by silica gel column (eluent: n-Hx:EA=20:1, v/v) to 26.1g of the target compound (yield: 85%) was obtained.
실시예 Example
2: 12: 1
-Conjugated--Conjugated-
linoeoyllineoyl
-2--2-
palmitoylpalmitoyl
-3-acetyl glycerol (CPA glycerol)의 제조Preparation of -3-acetyl glycerol (CPA glycerol)
(1) 1-C-linoleoyl glycerol의 제조(1) Preparation of 1-C-linoleoyl glycerol
n-Hexane 200ml에 conjugated Linoleic acid(cis-9,trans-11/trans-10,cis-12) 22.0g과 triethylamine 10.8g을 첨가하고 0℃로 냉각하고 Pivaloyl chloride 7.0g을 천천히 적가하고 동온도에서 1시간 동안 교반한 다음, Solketal 10.8g과 4-Dimethylaminopyridine 0.1g을 첨가한 후 실온에서 12시간 교반하고 정제수 200ml를 첨가하고 층분리 후 유기층은 진공 농축하였다. 농축 모액에 methanol 200ml, 염산 20ml를 투입하고 실온에서 교반하여 10시간 교반 후 n-hexane 300ml, 정제수 300ml를 투입하고 3시간 교반 후 여과하고 진공 건조하여 목적 화합물 22.8g (수율: 82%)을 수득하였다. 22.0 g of conjugated linoleic acid (cis-9,trans-11/trans-10,cis-12) and 10.8 g of triethylamine were added to 200 ml of n-Hexane, cooled to 0° C., and 7.0 g of Pivaloyl chloride was slowly added dropwise at the same temperature. After stirring for 1 hour, 10.8 g of Solketal and 0.1 g of 4-dimethylaminopyridine were added, followed by stirring at room temperature for 12 hours, 200 ml of purified water was added, and the organic layer was concentrated in vacuo after layer separation. 200ml of methanol and 20ml of hydrochloric acid were added to the concentrated mother liquor, stirred at room temperature, stirred for 10 hours, then added 300ml of n-hexane and 300ml of purified water, stirred for 3 hours, filtered and vacuum dried to obtain 22.8g of the target compound (yield: 82%). did.
(2) 1-C-linoleoyl-3-acetyl glycerol의 제조(2) Preparation of 1-C-linoleoyl-3-acetyl glycerol
MC 200ml에 1-C-linoleoyl glycerol 20.0g과 pyridine 33.5g, 4-Dimethylaminopyridine 0.2g을 첨가하고 1시간 교반한 다음, Acetyl chloride 6.6g을 적가하고 5시간 교반하였다. 정제수 200ml를 투입하고 묽은 염산으로 중화하고 층분리한 후 유기층을 MgSO4로 탈수한 후 진공 농축하고 n-hexane 100ml를 투입하고 10℃ 이하에서 결정화하여 여과 후 진공건조하여 목적 화합물 17.7g (수율: 79.0%)을 수득하였다.To 200 ml of MC, 20.0 g of 1-C-linoleoyl glycerol, 33.5 g of pyridine, and 0.2 g of 4-dimethylaminopyridine were added and stirred for 1 hour, then 6.6 g of acetyl chloride was added dropwise and stirred for 5 hours. After adding 200 ml of purified water, neutralizing with dilute hydrochloric acid, separating the layers , dehydrating the organic layer with MgSO 4 , vacuum concentration, adding 100 ml of n-hexane, crystallization at 10° C. or less, filtration, and vacuum drying the target compound 17.7 g (yield: 79.0%) was obtained.
(3) 1-C-linoleoyl-2-palmitoyl-3-acetyl glycerol (CPA glycerol)의 제조(3) Preparation of 1-C-linoleoyl-2-palmitoyl-3-acetyl glycerol (CPA glycerol)
n-Hexane 180ml에 Palmitic acid 12.2g과 triethylamine 10.8g을 첨가하고 0℃로 냉각하여 Pivaloyl chloride 7.0g을 천천히 적가하였다. 적가 완료 후 동온도에서 1시간 교반 후 1-C-linoleoyl-3-acetyl glycerol 18.0g과 4-Dimethylaminopyridine 1.0g을 첨가하고 실온에서 10시간 교반하였다. 정제수 180ml를 투입하고 층분리 하고 유기층은 MgSO4로 탈수한 후 진공 농축하고 실리카겔 컬럼 정제(용출액: n-Hx:EA=20:1, v/v)하여 목적화합물 25.9g (수율: 86%)을 수득하였다.12.2 g of palmitic acid and 10.8 g of triethylamine were added to 180 ml of n-Hexane, cooled to 0° C., and 7.0 g of pivaloyl chloride was slowly added dropwise. After completion of the dropwise addition, after stirring for 1 hour at the same temperature, 18.0 g of 1-C-linoleoyl-3-acetyl glycerol and 1.0 g of 4-dimethylaminopyridine were added and stirred at room temperature for 10 hours. 180ml of purified water was added, the layers were separated, and the organic layer was dehydrated with MgSO 4 , concentrated in vacuo, and purified by silica gel column (eluent: n-Hx:EA=20:1, v/v) to 25.9 g of the target compound (yield: 86%) was obtained.
비교예: 1-Palmitoyl-2-linoleoyl-3-acetyl glycerol (PLA glycerol)의 제조Comparative Example: Preparation of 1-Palmitoyl-2-linoleoyl-3-acetyl glycerol (PLA glycerol)
실시예 1와 동일한 방법으로 하되 conjugated Linoleic acid(cis-9,trans-11/trans-10,cis-12) 대신 Linoleic acid(cis-9,cis-12)를 사용하여 PLA glycerol을 합성하였다.PLA glycerol was synthesized in the same manner as in Example 1 using Linoleic acid (cis-9,cis-12) instead of conjugated Linoleic acid (cis-9,trans-11/trans-10,cis-12).
n-Hexane 180ml에 Linoleic acid(cis-9,cis-12) 14.0g과 triethylamine 10.8g을 첨가하고 0℃로 냉각하고 Pivaloyl chloride 7.0g을 천천히 적가하였다. 적가 완료 후 동온도에서 1시간 교반 후, 1-Palmitoyl-3-acetyl glycerol 18.0g과 4-Dimethylaminopyridine 1.0g을 첨가하고 실온에서 10시간 교반하였다. 정제수 180ml를 투입하고 층분리하고 유기층은 MgSO4로 탈수한 후 진공 농축하고 실리카겔 컬럼 정제(용출액: n-Hx:EA=20:1, v/v)하여 목적화합물 24.9g (수율: 81%)을 수득하였다.Linoleic acid (cis-9,cis-12) 14.0 g and triethylamine 10.8 g were added to 180 ml of n-Hexane, cooled to 0° C., and 7.0 g of pivaloyl chloride was slowly added dropwise. After completion of the dropwise addition, after stirring at the same temperature for 1 hour, 18.0 g of 1-Palmitoyl-3-acetyl glycerol and 1.0 g of 4-dimethylaminopyridine were added and stirred at room temperature for 10 hours. 180ml of purified water was added, the layers were separated, and the organic layer was dehydrated with MgSO 4 , concentrated in vacuo, and purified by silica gel column (eluent: n-Hx:EA=20:1, v/v) to 24.9g of the target compound (yield: 81%) was obtained.
상기 실시예 1, 2 및 비교예에서 합성된 화합물의 항염증 활성 실험을 다음과 같은 동물실험으로 평가하였다.The anti-inflammatory activity experiments of the compounds synthesized in Examples 1 and 2 and Comparative Examples were evaluated in the following animal experiments.
A. 실험동물A. Experimental animals
실험동물로서 8주령 C57BL/6 마우스를 온도 22±2℃, 상대습도 65±5%로, 명암 12시간 주기의 환경에서 7일 동안 사육하여 실험실 환경에 적응시킨 뒤 실험에 사용하였다. 고형사료(삼양사료)와 물을 충분히 공급하였다.As experimental animals, 8-week-old C57BL/6 mice were bred for 7 days at a temperature of 22±2° C., a relative humidity of 65±5%, and a light/dark cycle of 12 hours for 7 days, and then used for the experiment after adapting to the laboratory environment. Solid feed (Samyang feed) and water were sufficiently supplied.
B. 복강대식세포의 분리 배양B. Isolation of peritoneal macrophages
마우스에 HBSS를 복강 주사하여 대식세포(macrophage)를 추출하고, 3,000rpm에 5분간 원심분리 후 10% 소태아혈청(FBS)을 첨가한 DMEM배지에 100units/mL의 penicillin/streptomycin을 넣어 복강대식세포를 분리하였으며, 37℃, 5% CO2 배양기에서 24시간 배양후 실험에 사용하였다.HBSS was injected intraperitoneally into mice to extract macrophages, and after centrifugation at 3,000 rpm for 5 minutes, 100 units/mL of penicillin/streptomycin was added to DMEM medium supplemented with 10% fetal bovine serum (FBS). was isolated, and was used for the experiment after 24 hours of incubation in an incubator at 37° C., 5% CO 2
실험예 1: MTT assay를 통한 세포 독성 평가Experimental Example 1: Cytotoxicity evaluation through MTT assay
상기 배양된 복강대식세포를 3×105 cells/well로 96웰 플레이트에 100uL씩 분주하여 밤새 배양하였다. 배지를 제거한 후 상기 실시예 1(PCA glycerol), 실시예 2(CPA glycerol)의 화합물을 농도별(각각 10㎍/ml, 100㎍/ml 및 200㎍/ml)로 복강대식세포에 처리한 다음, 37℃, 5% CO2 배양기에서 24시간 동안 배양하였다. 배양 후 배지를 제거하고 MTT(5mg/mL) 시약을 40uL씩 분주하고 CO2 배양기에서 4시간 배양한 뒤, MTT 시약을 제거하고 DMSO 시약을 600uL씩 분주한 다음 30분간 상온에서 방치하였다. 이후 마이크로플레이트 판독기(microplate reader)로 540 nm에서 흡광도(OD)를 측정하였다.The cultured peritoneal macrophages were aliquoted at 3×10 5 cells/well in a 96-well plate by 100 uL and cultured overnight. After removing the medium, the compounds of Example 1 (PCA glycerol) and Example 2 (CPA glycerol) were treated in peritoneal macrophages by concentration (10㎍/ml, 100㎍/ml and 200㎍/ml, respectively), and then , 37° C., 5% CO 2 Incubated for 24 hours in an incubator. After incubation, the medium was removed, 40uL of MTT (5mg/mL) reagent was dispensed, and incubated for 4 hours in a CO 2 incubator, MTT reagent was removed, and DMSO reagent was dispensed 600uL each, and then left at room temperature for 30 minutes. Then, the absorbance (OD) was measured at 540 nm with a microplate reader.
실시예 1 및 실시예 2의 화합물의 MTT assay에 따른 세포 생존율(cell viability)을 측정하여 하기 표 2와 도 6의 그래프로 나타내었다. 표 2 및 도 6에 보이는 바와 같이 세포 생존율은 최고 200㎍/ml 처리시에도 유의적인 차이가 없음을 알 수 있다. 따라서, 본 발명에 따른 신규 화합물은 세포 독성이 없음이 확인되었다.Cell viability (cell viability) of the compounds of Examples 1 and 2 according to the MTT assay was measured and shown as a graph in Table 2 and FIG. 6 below. As shown in Table 2 and FIG. 6, it can be seen that there is no significant difference in cell viability even at the highest 200 μg/ml treatment. Therefore, it was confirmed that the novel compound according to the present invention is not cytotoxic.
| 정상군normal group | 실시예1 (㎍/ml)Example 1 (μg/ml) | 실시예2 (㎍/ml)Example 2 (μg/ml) | |||||
| 1010 | 100100 | 200200 | 1010 | 100100 | 200200 | ||
|
세포 생존율(%)cell Survival rate (%) |
100±1.2100±1.2 | 100±2.1100±2.1 | 98.3±1.998.3±1.9 | 98.1±1.298.1±1.2 | 100±0.9100±0.9 | 98.3±1.798.3±1.7 | 98.0±2.898.0±2.8 |
실험예 2: 항염증 활성 실험(IL-6, IL-1β 및 TNF-α 생성 억제 평가)Experimental Example 2: Anti-inflammatory activity test (IL-6, IL-1β and TNF-α production inhibition evaluation)
염증 반응 유도인자인 지질다당류(lipopolysaccharides, LPS)로 유도된 마우스 복강대식세포로부터 분비된 IL-6, IL-1β 및 TNF-α 분비량을 ELISA assay(Millipore사, USA)를 이용하여 측정함으로써 항염증 활성을 평가하였다.Anti-inflammatory by measuring the secretion of IL-6, IL-1β and TNF-α from mouse peritoneal macrophages induced by lipopolysaccharides (LPS), an inflammatory response inducer, using an ELISA assay (Millipore, USA). Activity was assessed.
세포 배양액을 얻기 위해 마우스 복강대식세포를 3×105 cells/mL로 조절하여 96웰 플레이트에 접종하고 24시간 배양 후 상기 실시예 1(PCA glycerol), 실시예 2(CPA glycerol) 및 비교예(PLA glycerol)의 화합물을 농도별(각각 10㎍/ml, 100㎍/ml 및 200㎍/ml)로 처리하고, LPS(1㎍/ml)를 처리하였다. 정상군는 무처리, 대조군은 LPS(1㎍/ml)만을 복강대식세포에 처리하였다. 12시간 배양 후 원심분리를 통해 상층액을 얻었다. ELISA는 마이크로플레이트에 포획 항체(capture antibody)로 항-마우스 IL-6, IL-1β 및 TNF-α를 분주하였다. 이후 0.05% Tween 20이 포함된 인산완충식염수(PBST)로 세척하고 10% FBS으로 차단한 뒤 PBST로 세척하고 웰에 세포 배양 상층액을 분주하고 실온에서 2시간 반응시켰다. 반응 후 PBST로 세척하고 희석한 비오티닐된(biotinylated) 항-마우스 IL-6, IL-1β 및 TNF-α 검출 항체(detection antibody)와 스트렙타비딘-HRP 접합체(streptavidin-horseradish peroxydase conjugate)를 분주하여 실온에서 1시간 반응시켰다. 그 후 다시 PBST로 세척하고 OPD 용액을 첨가하여 실온에서 30분 동안 암반응시켰다. 2 N H2SO4로 반응을 종료시킨 후 마이크로플레이트 리더(microplate reader)를 이용하여 450 nm에서 흡광도를 측정하였다.In order to obtain a cell culture solution, mouse peritoneal macrophages were adjusted to 3 × 10 5 cells/mL, inoculated in a 96-well plate, and cultured for 24 hours in Example 1 (PCA glycerol), Example 2 (CPA glycerol) and Comparative Example ( PLA glycerol) was treated with each concentration (10 μg/ml, 100 μg/ml and 200 μg/ml, respectively), and LPS (1 μg/ml) was treated. The normal group was untreated, and the control group was treated with only LPS (1 μg/ml) in abdominal macrophages. After 12 hours of incubation, the supernatant was obtained by centrifugation. For ELISA, anti-mouse IL-6, IL-1β and TNF-α were dispensed on microplates as capture antibodies. Then, it was washed with phosphate buffered saline (PBST) containing 0.05% Tween 20, blocked with 10% FBS, washed with PBST, and the cell culture supernatant was dispensed into the wells and reacted for 2 hours at room temperature. After the reaction, biotinylated anti-mouse IL-6, IL-1β, and TNF-α detection antibody and streptavidin-HRP conjugate (streptavidin-horseradish peroxydase conjugate) were washed and diluted with PBST It was aliquoted and reacted at room temperature for 1 hour. After that, it was washed again with PBST, and an OPD solution was added, followed by dark reaction at room temperature for 30 minutes. After terminating the reaction with 2 NH 2 SO 4 , the absorbance was measured at 450 nm using a microplate reader.
실시예 1 및 실시예 2의 화합물의 IL-6 생성량을 측정하여 하기 표 3에 도시하고, 도 7의 그래프로 나타내었다. 표 3 및 도 7에 보이는 바와 같이 실시예 1 및 2에서 제조된 화합물은 대조군에 비해 IL-6 생성량이 현저하게 감소되었으며, 비교예와 대비할 때도 유의적인 IL-6 생성 억제 효과를 가지는 것을 알 수 있다.The IL-6 production amount of the compounds of Examples 1 and 2 was measured and shown in Table 3 below, and is shown in the graph of FIG. 7 . As shown in Table 3 and Figure 7, it can be seen that the compounds prepared in Examples 1 and 2 significantly reduced IL-6 production compared to the control, and had a significant IL-6 production inhibitory effect even when compared to Comparative Examples there is.
| LPS (1㎍/ml)LPS (1㎍/ml) | |||||||||||
| 정상군normal group | 대조군control | 실시예1 (㎍/ml)Example 1 (μg/ml) | 실시예2 (㎍/ml)Example 2 (μg/ml) | 비교예 (㎍/ml)Comparative Example (μg/ml) | |||||||
| 1010 | 100100 | 200200 | 1010 | 100100 | 200200 | 1010 | 100100 | 200200 | |||
|
IL-6 생성량 (pg/ml)IL-6 amount of production (pg/ml) |
150±18150±18 | 834±32834±32 | 321±22321±22 | 225±23225±23 | 180±25180±25 | 325±13325±13 | 234±20234±20 | 179±17179±17 | 364±16364±16 | 251±24251±24 | 203±25203±25 |
실시예 1 및 실시예 2의 화합물의 IL-1β 생성량을 측정하여 하기 표 4에 도시하고, 도 8의 그래프로 나타내었다. 표 4 및 도 8에 보이는 바와 같이 실시예 1 및 2에서 제조된 화합물은 대조군에 비해 IL-1β 생성량이 현저하게 감소되었으며, 비교예와 대비할 때도 유의적인 IL-1β 생성 억제 효과를 가지는 것을 알 수 있다.The IL-1β production amount of the compounds of Examples 1 and 2 was measured and shown in Table 4 below, and is shown in the graph of FIG. 8 . As shown in Tables 4 and 8, the compounds prepared in Examples 1 and 2 significantly reduced the amount of IL-1β production compared to the control, and it can be seen that they also had a significant IL-1β production inhibitory effect when compared with the comparative example. there is.
| LPS (1㎍/ml)LPS (1㎍/ml) | |||||||||||
| 정상군normal group | 대조군control | 실시예1 (㎍/ml)Example 1 (μg/ml) | 실시예2 (㎍/ml)Example 2 (μg/ml) | 비교예 (㎍/ml)Comparative Example (μg/ml) | |||||||
| 1010 | 100100 | 200200 | 1010 | 100100 | 200200 | 1010 | 100100 | 200200 | |||
|
IL-1β 생성량 (pg/ml) IL-1β amount of production (pg/ml) |
25±1.825±1.8 | 72±3.572±3.5 | 40±2.940±2.9 | 28±2.328±2.3 | 26±2.826±2.8 | 40±1.740±1.7 | 32±2.432±2.4 | 28±1.728±1.7 | 43±2.243±2.2 | 31±2.431±2.4 | 31±3.131±3.1 |
실시예 1 및 실시예 2의 화합물의 TNF-α 생성량을 측정하여 하기 표 5에 도시하고, 도 9의 그래프로 나타내었다. 표 5 및 도 9에 보이는 바와 같이 실시예 1 및 2에서 제조된 화합물은 대조군에 비해 TNF-α 생성량이 현저하게 감소되었으며, 비교예와 대비할 때도 유의적인 TNF-α 생성 억제 효과를 가지는 것을 알 수 있다.The TNF-α production amount of the compounds of Examples 1 and 2 was measured and shown in Table 5 below, and is shown in the graph of FIG. 9 . As shown in Table 5 and Figure 9, the compounds prepared in Examples 1 and 2 significantly reduced the amount of TNF-α production compared to the control, and it can be seen that also has a significant TNF-α production inhibitory effect when compared with the comparative example. there is.
| LPS(1㎍/ml)LPS (1 μg/ml) | |||||||||||
| 정상군normal group | 대조군control | 실시예1 (㎍/ml)Example 1 (μg/ml) | 실시예2 (㎍/ml)Example 2 (μg/ml) | 비교예 (㎍/ml)Comparative Example (μg/ml) | |||||||
| 1010 | 100100 | 200200 | 1010 | 100100 | 200200 | 1010 | 100100 | 200200 | |||
|
TNF-α 생성량 (pg/ml)TNF-α production (pg/ml) |
113±11113±11 | 375±16375±16 | 170±8170±8 | 153±11153±11 | 121±10121±10 | 175±7175±7 | 151±10151±10 | 123±7123±7 | 184±21184±21 | 155±12155±12 | 137±7137±7 |
실험예 3: 항염증 활성 실험(NO 형성 억제 평가)Experimental Example 3: Anti-inflammatory activity test (NO formation inhibition evaluation)
상기 배양된 복강대식세포를 10% FBS가 포함된 DMEM에 현탁시킨 후, 96웰 플레이트에 5×105 cells/well로 분주하여 37℃, 5% CO2 배양기에서 24시간 배양하고, 새로운 DMEM배지로 교환한 후, 실시예 1, 2 및 비교예의 화합물 각각을 농도별(각각 10㎍/ml, 100㎍/ml 및 200㎍/ml)로 복강대식세포에 처리하고 LPS(1㎍/ml)를 처리한 다음, 24시간 동안 배양하였다. 배양 후 상층액을 분리하여 3000 rpm에서 5분간 원심분리하여 분리된 상층액을 새로운 마이크로플레이트(microplate)에 분주하였다. 정상군는 무처리, 대조군은 LPS(1㎍/ml)만을 복강대식세포에 처리하였다.The cultured peritoneal macrophages were suspended in DMEM containing 10% FBS, and then aliquoted at 5×10 5 cells/well in a 96-well plate and cultured at 37° C., 5% CO 2 in an incubator for 24 hours, and fresh DMEM medium. After exchanging with peritoneal macrophages, each of the compounds of Examples 1 and 2 and Comparative Example was treated with each concentration (10㎍/ml, 100㎍/ml and 200㎍/ml, respectively) in peritoneal macrophages and LPS (1㎍/ml) After treatment, incubated for 24 hours. After incubation, the supernatant was separated and centrifuged at 3000 rpm for 5 minutes, and the separated supernatant was dispensed into a new microplate. The normal group was untreated, and the control group was treated with only LPS (1 μg/ml) in abdominal macrophages.
동일한 양의 Griess 시약(1% sulfanilamide, 0.1% naphthyl-ethylenediamine dihydrochloride, 2% phosphoric acid)을 처리하여 상온에서 10분 반응시켰다. 배양액과 Griess 용액을 5분 동안 반응시킨 후 540nm에서 흡광도(OD)를 측정하였다.The same amount of Griess reagent (1% sulfanilamide, 0.1% naphthyl-ethylenediamine dihydrochloride, 2% phosphoric acid) was treated and reacted at room temperature for 10 minutes. After reacting the culture medium with the Griess solution for 5 minutes, absorbance (OD) was measured at 540 nm.
도 10은 본 발명의 실시예 1 및 실시예 2의 신규 화합물의 NO 생성 억제 효과를 나타내는 그래프이다.10 is a graph showing the NO production inhibitory effect of the novel compounds of Examples 1 and 2 of the present invention.
실시예 1 및 실시예 2의 화합물의 일산화질소(NO) 생성율을 측정하여 하기 표 6에 도시하고, 도 10의 그래프로 나타내었다. 표 6 및 도 10에 보이는 바와 같이 실시예 1 및 2에서 제조된 화합물은 대조군에 비해 NO 생성율이 현저하게 감소되었으며, 비교예와 대비할 때도 유의적인 NO 생성 억제 효과를 가지는 것을 알 수 있다.The nitrogen monoxide (NO) production rates of the compounds of Examples 1 and 2 were measured and shown in Table 6 below, and shown in the graph of FIG. 10 . As shown in Tables 6 and 10, the compounds prepared in Examples 1 and 2 significantly reduced the NO production rate compared to the control, and it can be seen that they have a significant NO production inhibitory effect even when compared to the comparative example.
| LPS (㎍/ml)LPS (μg/ml) | |||||||||||
| 정상군normal group | 대조군control | 실시예1 (㎍/ml)Example 1 (μg/ml) | 실시예2 (㎍/ml)Example 2 (μg/ml) | 비교예 (㎍/ml)Comparative Example (μg/ml) | |||||||
| 1010 | 100100 | 200200 | 1010 | 100100 | 200200 | 1010 | 100100 | 200200 | |||
|
NO 생성율 (%)NO production rate (%) |
25±225±2 | 100±4100±4 | 52±352±3 | 43±343±3 | 37±437±4 | 50±250±2 | 44±344±3 | 34±134±1 | 56±356±3 | 45±245±2 | 41±341±3 |
실험예 4: 만성폐쇄성 폐질환(COPD) 동물모델 실험Experimental Example 4: Chronic Obstructive Pulmonary Disease (COPD) Animal Model Experiment
만성폐쇄성 폐질환에 대한 실시예 1 및 실시예 2에서 합성된 화합물의 약학적 효과를 확인하기 위하여 동물실험을 진행하였다. 만성폐쇄성 폐질환은 담배연기 추출물(Cigarette smoking soulution, CSS)을 마우스의 폐에 주입하여 유발시켰다.Animal experiments were conducted to confirm the pharmaceutical effects of the compounds synthesized in Examples 1 and 2 on chronic obstructive pulmonary disease. Chronic obstructive pulmonary disease was induced by injecting Cigarette smoking solution (CSS) into the lungs of mice.
(A) 담배연기 추출물(Cigarette smoking soulution, CSS)의 준비(A) Preparation of Cigarette smoking soulution (CSS)
ISO 3308 규정에 따른 자동흡연장치(RM20/CS, Heinrich Borgwaldt사, 독일)를 이용하여 실험용 표준담배인 Coresta Monitoring Cigarette No. 7(CM7, Heinrich Borgwaldt사, 독일)을 연소시키고, 92mm 캠브리지 필터(cambridge filter)에 담배연기 응축물을 포집하였다.Using an automatic smoking device (RM20/CS, Heinrich Borgwaldt, Germany) in accordance with ISO 3308 regulations, Coresta Monitoring Cigarette No. 7 (CM7, Heinrich Borgwaldt, Germany) was burned, and tobacco smoke condensate was collected in a 92 mm Cambridge filter.
담배연기 응축물의 포집은 ISO 3402 규정에 따라 온도 22±℃, 상대습도 60±5% 조건의 흡연실에서 실시하였고, 흡연방법은 ISO 3308 규정에 따라 3mm 이상의 꽁초길이, 35.0±0.3ml의 흡연부피, 2.00±0.02초의 흡연시간, 60±0.5초의 흡연주기 조건에서 실시하였다.Tobacco smoke condensate was collected in a smoking room with a temperature of 22±℃ and a relative humidity of 60±5% according to ISO 3402 regulations, and the smoking method was a butt length of 3mm or more, a smoking volume of 35.0±0.3ml according to ISO 3308 regulations; It was carried out under the conditions of a smoking time of 2.00±0.02 seconds and a smoking cycle of 60±0.5 seconds.
담배연기 응축물이 포집된 캠브리지 필터(cambridge filter)를 이소프로판올에 넣고 흔든 다음, 실온에서 8시간 이상을 방치하여 추출, 여과한 다음, 감압여과 농축기 및 질소 가스를 이용하여 완전 농축하여 표준담배 추출물(CSS)을 제조하였다.A Cambridge filter in which the cigarette smoke condensate is collected is placed in isopropanol and shaken, left at room temperature for more than 8 hours, extracted and filtered, and then completely concentrated using a vacuum filtration concentrator and nitrogen gas to extract a standard tobacco extract ( CSS) was prepared.
(B) 만성폐쇄성 폐질환(COPD) 모델 제작(B) Chronic obstructive pulmonary disease (COPD) model production
8주령 C57BL/6 마우스를 대상으로 LPS 100㎍/ml와 표준담배 추출물(CSS) 4mg/ml을 1:1로 혼합하여 COPD 유발물질(LPS+CSS)을 제조하고, 주 1회씩 3주간 코와 입을 통하여 유발물질 50㎕씩 총 100㎕를 흡인시켜 COPD를 유발시켰다. 흡인은 7% 클로랄 수화물(Chloral hydrate)을 복강 주사하여 약간 마취시켜 움직임이 없을 때 생쥐의 앞니를 고무밴드로 고정시킨 상태에서 기관내 투여하였다.For 8-week-old C57BL/6 mice, 100 μg/ml of LPS and 4 mg/ml of standard tobacco extract (CSS) were mixed 1:1 to prepare COPD-inducing substances (LPS+CSS), and once a week for 3 weeks COPD was induced by aspirating a total of 100 μl of each 50 μl of the inducer through the mouth. For aspiration, 7% chloral hydrate was injected intraperitoneally to slightly anesthetize, and when there was no movement, the incisors of the mice were fixed with a rubber band and administered intratracheally.
실시예 1의 화합물, 실시예 2의 화합물, 비교예의 화합물은 각각 60 mg/kg을 0.5% CMC(carboxmethylcellulose sodium)에 녹여, 유발물질 기관 흡입 1시간 전에 미리 경구투여하였다. 실험군은 (i)아무런 처리를 하지 않은 정상군(Normal), (ii)유발물질(LPS+CSS)을 처리한 대조군(Control), (iii)유발물질 처리 1시간 전 실시예 1의 화합물을 경구투여한 투여군, (iv) 유발물질 처리 1시간 전 실시예 2의 화합물을 경구투여한 투여군, (v)유발물질 처리 1시간 전 비교예의 화합물을 경구투여한 투여군으로 하였다. 실험 종료 후 채혈한 후, 개흉하여 기도를 노출시켜 FBS free DMEM 배지 1㎖을 넣은 주사기를 기관지(trachea)에 삽입하여 주입시키고, 끈으로 묶어 고정한 후에 기관지 폐포세척을 3회 순환하여 세척액(BALF)을 얻었다. Each of the compound of Example 1, the compound of Example 2, and the compound of Comparative Example was dissolved in 60 mg/kg of 0.5% CMC (carboxmethylcellulose sodium) and orally administered 1 hour before inhalation of the inducer organ. The experimental group was (i) a normal group without any treatment (Normal), (ii) a control group treated with an inducer (LPS+CSS), (iii) the compound of Example 1 was orally administered 1 hour before the trigger material treatment The administration group was administered, (iv) the administration group in which the compound of Example 2 was orally administered 1 hour before the inducer treatment, and (v) the administration group in which the compound of Comparative Example was orally administered 1 hour before the inducer substance treatment. After the end of the experiment, blood was collected, open the chest, expose the airways, insert a syringe containing 1 ml of FBS-free DMEM medium into the trachea, and fix it with a string, then circulate bronchoalveolar lavage 3 times to obtain lavage solution (BALF). got
실험예 5: 기관지 폐포세척액(BALF) 내 총 호중구 세포수 측정Experimental Example 5: Measurement of total neutrophil cell count in bronchoalveolar lavage (BALF)
얻어진 BALF를 4℃, 2,000 rpm, 5분간 원심분리하여 침전된 혈구를 분리한 다음, Diff-Quick Stain(Romanowsky stain)을 3회에 걸쳐 처리하고, 이후 PBS로 2회 세척한 후 군당 9개의 슬라이드를 제작하여 400배율의 광학현미경을 통해 계수하여 하기 표 7 및 도 11에 도시하였다.The obtained BALF was centrifuged at 4°C, 2,000 rpm, for 5 minutes to separate the precipitated blood cells, then treated with Diff-Quick Stain (Romanowsky stain) three times, and then washed twice with PBS, and then 9 slides per group was produced and counted through an optical microscope at 400 magnification, and is shown in Table 7 and FIG. 11 below.
| -- | LPS+CSSLPS+CSS | ||||
| 정상군normal group | 대조군control | 실시예1Example 1 | 실시예2Example 2 | 비교예comparative example | |
| 호중구 수(/㎕)Neutrophil count (/μl) | 11.1±1.611.1±1.6 | 231.2±14.5231.2±14.5 | 73.4±21.673.4±21.6 | 82.0±18.982.0±18.9 | 122.7±27.4122.7±27.4 |
| 억제율(%)Inhibition rate (%) | -- | -- | 68%68% | 65%65% | 47%47% |
상기 표 7 및 도 11에 나타나는 바와 같이, COPD를 유발한 대조군에서 호중구는 231.2±14.5개로 나타나 정상군의 11.1±1.6개보다 현저히 증가하였으나, 실시예 1의 화합물 투여군은 73.4±21.6개로 대조군 대비 68% 호중구 억제율, 실시예 2의 화합물 투여군은 82.0±18.9개로 65% 호중구 억제율로 비교예(47% 호중구 억제율)와 비교할 때에는 호중구 수에 있어 유의적인 감소 효과가 있음이 확인되었다.실험예 6: BALF 내 사이토카인 분비량 측정(COPD 억제 실험)
As shown in Table 7 and FIG. 11, the number of neutrophils in the COPD-induced control group was 231.2±14.5, which was significantly increased compared to 11.1±1.6 in the normal group, but the compound administered group of Example 1 had 73.4±21.6, 68 compared to the control group. % neutrophil inhibition, the compound administered group of Example 2 was 82.0±18.9, and it was confirmed that there was a significant reduction effect on the number of neutrophils when compared with the comparative example (47% neutrophil inhibition) with a 65% neutrophil inhibition rate. Experimental Example 6: Measurement of cytokine secretion in BALF (COPD inhibition experiment)
만성폐쇄성 폐질환(COPD) 환자의 BALF 내에는 TNF-α, MIP2, CXCL-1, CXCL-8, IL-1β, IL-6 등의 다양한 사이토카인이 증가되는 것으로 알려져 있다.It is known that various cytokines such as TNF-α, MIP2, CXCL-1, CXCL-8, IL-1β, and IL-6 are increased in the BALF of chronic obstructive pulmonary disease (COPD) patients.
실험예 4에서 얻어진 BALF 내 TNF-α, MIP2, CXCL1 분비량을 ELISA assay(Millipore사, USA)를 이용하여 측정하여 COPD 억제 효과를 평가하였다.The amount of TNF-α, MIP2, and CXCL1 secretion in BALF obtained in Experimental Example 4 was measured using an ELISA assay (Millipore, USA) to evaluate the COPD inhibitory effect.
항-마우스 TNF-α, MIP2 및 CXCL-1 포획 항체(capture antibody)를 마이크로플레이트에 분주하였다. 이후 0.05% Tween 20이 포함된 인산완충식염수(PBST)로 세척하고 10% FBS으로 차단한 뒤 PBST로 세척하고 웰에 BAL 세포를 분주하고 실온에서 2시간 반응시켰다. 반응 후 PBST로 세척하고 희석한 비오티닐된(biotinylated) 항-마우스 TNF-α, MIP2 및 CXCL-1 검출 항체(detection antibody)와 스트렙타비딘-HRP 접합체(streptavidin-horseradish peroxydase conjugate)를 분주하여 실온에서 1시간 반응시켰다. 그 후 다시 PBST로 세척하고 OPD 용액을 첨가하여 실온에서 30분 동안 암반응시켰다. 2N H2SO4로 반응을 종료시킨 후 마이크로플레이트 리더(microplate reader)를 이용하여 450 nm에서 흡광도를 측정하였다.Anti-mouse TNF-α, MIP2 and CXCL-1 capture antibodies were aliquoted into microplates. Then, it was washed with phosphate buffered saline (PBST) containing 0.05% Tween 20, blocked with 10% FBS, washed with PBST, and BAL cells were seeded in the wells and reacted for 2 hours at room temperature. After the reaction, biotinylated anti-mouse TNF-α, MIP2, and CXCL-1 detection antibodies and streptavidin-HRP conjugate (streptavidin-horseradish peroxydase conjugate) that were washed and diluted with PBST were dispensed. The reaction was carried out at room temperature for 1 hour. After that, it was washed again with PBST, and an OPD solution was added, followed by dark reaction at room temperature for 30 minutes. After terminating the reaction with 2N H 2 SO 4 , the absorbance was measured at 450 nm using a microplate reader.
실시예 1 및 실시예 2의 화합물의 TNF-α, MIP2 및 CXCL-1 생성량을 각각 측정하여 하기 표 8 내지 표 10에 도시하고, 도 12 내지 14의 그래프로 나타내었다.The TNF-α, MIP2 and CXCL-1 production amounts of the compounds of Examples 1 and 2 were measured, respectively, and shown in Tables 8 to 10 below, and shown in the graphs of FIGS. 12 to 14 .
| -- | LPS+CSSLPS+CSS | ||||
| 정상군normal group | 대조군control | 실시예1Example 1 | 실시예2Example 2 | 비교예comparative example | |
|
TNF-α 생성량 (pg/ml)TNF-α amount of production (pg/ml) |
7.1±1.17.1±1.1 | 151.6±10.6151.6±10.6 | 42.3±8.542.3±8.5 | 39.8±6.739.8±6.7 | 58.4±7.158.4±7.1 |
| 억제율(%)Inhibition rate (%) | -- | -- | 72%72% | 74%74% | 61%61% |
상기 표 8 및 도 12에 나타나는 바와 같이, COPD를 유발한 대조군에서 TNF-α 생성량은 151.6±10.6 pg/ml로 나타나 정상군의 7.1±1.1 pg/ml보다 현저히 증가하였으나, 실시예 1의 화합물 투여군은 42.3±8.5 pg/ml로 대조군 대비 72% 억제율, 실시예 2의 화합물 투여군은 39.8±6.7 pg/ml로 74% 억제율로 비교예(61% 억제율)와 비교할 때 TNF-α 생성량에 있어 유의적인 감소 효과가 있음이 확인되었다.As shown in Table 8 and FIG. 12, the amount of TNF-α production in the COPD-induced control group was 151.6±10.6 pg/ml, which was significantly increased compared to 7.1±1.1 pg/ml in the normal group, but the compound administered group of Example 1 is 42.3±8.5 pg/ml, 72% inhibition compared to the control group, and the compound administered group of Example 2 was 39.8±6.7 pg/ml, 74% inhibition, which was significant in TNF-α production compared to the comparative example (61% inhibition). It was confirmed that there is a reduction effect.
| -- | LPS+CSSLPS+CSS | ||||
| 정상군normal group | 대조군control | 실시예1Example 1 | 실시예2Example 2 | 비교예comparative example | |
|
MIP2 생성량 (pg/ml)MIP2 amount of production (pg/ml) |
62.1±7.162.1±7.1 | 301.4±16.7301.4±16.7 | 91.2±11.591.2±11.5 | 95.3±5.695.3±5.6 | 111.4±8.9111.4±8.9 |
| 억제율(%)Inhibition rate (%) | -- | -- | 70%70% | 68%68% | 63%63% |
상기 표 9 및 도 13에 나타나는 바와 같이, COPD를 유발한 대조군에서 MIP2 생성량은 301.4±16.7 pg/ml로 나타나 정상군의 62.1±7.1 pg/ml보다 현저히 증가하였으나, 실시예 1의 화합물 투여군은 91.2±11.5 pg/ml로 대조군 대비 70% 억제율, 실시예 2의 화합물 투여군은 95.3±5.6 pg/ml로 68% 억제율로 비교예(63% 억제율)와 비교할 때 MIP2 생성량에 있어 유의적인 감소 효과가 있음이 확인되었다.As shown in Table 9 and FIG. 13, the amount of MIP2 production in the COPD-induced control group was 301.4±16.7 pg/ml, which was significantly increased compared to 62.1±7.1 pg/ml in the normal group, but the compound administered group of Example 1 was 91.2 70% inhibition rate compared to the control group at ±11.5 pg/ml, the compound administered group of Example 2 had a significant reduction effect in MIP2 production compared to the comparative example (63% inhibition rate) with a 68% inhibition rate at 95.3±5.6 pg/ml This was confirmed.
| -- | LPS+CSSLPS+CSS | ||||
| 정상군normal group | 대조군control | 실시예1Example 1 | 실시예2Example 2 | 비교예comparative example | |
|
CXCL-1 생성량 (pg/ml)CXCL-1 amount of production (pg/ml) |
64.2±8.164.2±8.1 | 250.4±18.7250.4±18.7 | 77.3±12.077.3±12.0 | 84.9±8.684.9±8.6 | 89.1±11.389.1±11.3 |
| 억제율(%)Inhibition rate (%) | -- | -- | 69%69% | 66%66% | 64%64% |
상기 표 10 및 도 14에 나타나는 바와 같이, COPD를 유발한 대조군에서 CXCL-1 생성량은 250.4±18.7 pg/ml로 나타나 정상군의 64.2±8.1 pg/ml보다 현저히 증가하였으나, 실시예 1의 화합물 투여군은 77.3±12.0 pg/ml로 대조군 대비 69% 억제율, 실시예 2의 화합물 투여군은 84.9±8.6 pg/ml로 66% 억제율로 비교예(64% 억제율)와 비교할 때 CXCL-1 생성량에 있어 유의적인 감소 효과가 있음이 확인되었다.본 발명에서 상기 화학식 1 내지 4의 화합물은 약학적 조성물의 유효성분으로 포함될 수 있다.As shown in Table 10 and FIG. 14, the amount of CXCL-1 produced in the control group induced by COPD was 250.4±18.7 pg/ml, which was significantly increased compared to 64.2±8.1 pg/ml in the normal group, but the group administered with the compound of Example 1 is 77.3±12.0 pg/ml, with a 69% inhibition rate compared to the control group, and the compound administered group of Example 2 was 84.9±8.6 pg/ml with a 66% inhibition rate, which was significant in the amount of CXCL-1 production compared to the comparative example (64% inhibition rate). It was confirmed that there is a reducing effect. In the present invention, the compounds of Formulas 1 to 4 may be included as an active ingredient in a pharmaceutical composition.
본 발명에서 상기 약학적 조성물은 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결 건조제 등 통상의 약학적 조성물 형태일 수 있으며, 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제 등 약학적 제제에 이용되는 첨가물들이 포함될 수 있다.In the present invention, the pharmaceutical composition is in the form of a conventional pharmaceutical composition such as tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-drying agents, etc. and may be in various oral or parenteral formulations. In the case of formulation, additives used in pharmaceutical formulations such as diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be included.
본 발명의 조성물은 약학적으로 유효한 양으로 투여할 수 있다. 본 발명에서 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 질병의 종류, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다.The composition of the present invention can be administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the subject's type and severity, age, sex, and disease. The type, activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple.
본 발명에서 상기 화학식 1 내지 4의 화합물은 건강기능식품의 유효성분으로 포함될 수 있다.In the present invention, the compounds of Formulas 1 to 4 may be included as an active ingredient of a health functional food.
본 발명에서 건강기능식품은 비의약품으로 사용되는 식품으로, 식품의 종류에는 특별한 제한은 없으며, 예시적으로는 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결 건조제 등 통상의 약학적 조성물 형태일 수 있으며, 식품 첨가물일 수 있다.In the present invention, health functional food is a food used as a non-pharmaceutical, and there is no particular limitation on the type of food, for example, tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilization. It may be in the form of a conventional pharmaceutical composition such as an aqueous solution, a non-aqueous solvent, a suspension, an emulsion, and a freeze-drying agent, and may be a food additive.
본 발명은 녹용에서 분리한 신규 화합물을 유효성분으로 하는 만성폐쇄성 폐질환의 예방 또는 치료용 약학적 조성물 또는 건강기능식품에 관한 것이다.The present invention relates to a pharmaceutical composition or health functional food for the prevention or treatment of chronic obstructive pulmonary disease, comprising a novel compound isolated from deer antler as an active ingredient.
Claims (6)
- 하기 화학식 1 내지 4로 표시되는 화합물에서 선택되는 1종 이상을 유효성분으로 하는, 만성폐쇄성 폐질환의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for the prevention or treatment of chronic obstructive pulmonary disease, comprising at least one selected from the compounds represented by the following formulas 1 to 4 as an active ingredient.[화학식 1][Formula 1]
[화학식 2][Formula 2]
[화학식 3][Formula 3]
[화학식 4][Formula 4]
- 제1항에 있어서,According to claim 1,상기 유효성분은 화학식 1로 표시되는 화합물 및 화학식 2로 표시되는 화합물의 혼합물인, 만성폐쇄성 폐질환의 예방 또는 치료용 약학적 조성물.The active ingredient is a mixture of the compound represented by Formula 1 and the compound represented by Formula 2, a pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease.
- 제1항에 있어서,According to claim 1,상기 유효성분은 화학식 3으로 표시되는 화합물 및 화학식 4로 표시되는 화합물의 혼합물인, 만성폐쇄성 폐질환의 예방 또는 치료용 약학적 조성물.The active ingredient is a mixture of the compound represented by Formula 3 and the compound represented by Formula 4, a pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease.
- 제1항에 있어서,According to claim 1,상기 만성폐쇄성 폐질환은 만성 기관지염 또는 폐기종인 것인, 만성폐쇄성 폐질환의 예방 또는 치료용 약학적 조성물.The chronic obstructive pulmonary disease is chronic bronchitis or emphysema, a pharmaceutical composition for the prevention or treatment of chronic obstructive pulmonary disease.
- 제1항에 있어서,According to claim 1,상기 유효성분은 폐에서 사이토카인의 생성을 억제하는 것인, 만성폐쇄성 폐질환의 예방 또는 치료용 약학적 조성물.The active ingredient is to inhibit the production of cytokines in the lung, a pharmaceutical composition for the prevention or treatment of chronic obstructive pulmonary disease.
- 하기 화학식 1 내지 4로 표시되는 화합물을 유효성분으로 포함하는, 만성폐쇄성 폐질환의 예방 또는 개선용 건강기능식품.A health functional food for preventing or improving chronic obstructive pulmonary disease, comprising a compound represented by the following Chemical Formulas 1 to 4 as an active ingredient.[화학식 1][Formula 1]
[화학식 2][Formula 2]
[화학식 3][Formula 3]
[화학식 4][Formula 4]
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020200008958A KR20210094996A (en) | 2020-01-22 | 2020-01-22 | Pharmaceuticals or health functional foods for treating or preventing Chronic Obstructive Pulmonary Disease comprising novel compounds isolated from Cervus nippon |
| KR10-2020-0008958 | 2020-01-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2021150076A1 true WO2021150076A1 (en) | 2021-07-29 |
Family
ID=76992903
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2021/000939 WO2021150076A1 (en) | 2020-01-22 | 2021-01-22 | Pharmaceutical composition or health functional food for preventing or treating chronic obstructive pulmonary diseases |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR20210094996A (en) |
| WO (1) | WO2021150076A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3824886A4 (en) * | 2018-07-16 | 2022-04-20 | FNG Research Co., Ltd. | Novel compounds isolated from cervi parvum cornu, and pharmaceutical uses thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080193550A1 (en) * | 2004-05-11 | 2008-08-14 | Fonterra Corporate Research And Development Limited | Cla-Enriched Milkfat and Uses Thereof |
| KR20150021472A (en) * | 2013-08-19 | 2015-03-02 | 한국생명공학연구원 | Composition for preventing or treating chronic obstructive pulmonary diseases comprising monoacetyldiacylglycerol compound |
| KR20150091715A (en) * | 2014-02-03 | 2015-08-12 | 최원형 | Anti-tuberculosis composition for treating or preventing tuberculosis comprising Artesunate or gamma-Linolenic acid |
-
2020
- 2020-01-22 KR KR1020200008958A patent/KR20210094996A/en unknown
-
2021
- 2021-01-22 WO PCT/KR2021/000939 patent/WO2021150076A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080193550A1 (en) * | 2004-05-11 | 2008-08-14 | Fonterra Corporate Research And Development Limited | Cla-Enriched Milkfat and Uses Thereof |
| KR20150021472A (en) * | 2013-08-19 | 2015-03-02 | 한국생명공학연구원 | Composition for preventing or treating chronic obstructive pulmonary diseases comprising monoacetyldiacylglycerol compound |
| KR20150091715A (en) * | 2014-02-03 | 2015-08-12 | 최원형 | Anti-tuberculosis composition for treating or preventing tuberculosis comprising Artesunate or gamma-Linolenic acid |
Non-Patent Citations (2)
| Title |
|---|
| JAUDSZUS, A. ; FOERSTER, M. ; KROEGEL, C. ; WOLF, I. ; JAHREIS, G.: "Cis-9,Trans-11-CLA exerts anti-inflammatory effects in human bronchial epithelial cells and eosinophils: Comparison to Trans-10,Cis-12-CLA and to linoleic acid", BIOCHIMICA ET BIOPHYSICA ACTA (BBA) - MOLECULAR AND CELL BIOLOGY OF LIPIDS, ELSEVIER, AMSTERDAM, NL, vol. 1737, no. 2-3, 15 December 2005 (2005-12-15), AMSTERDAM, NL, pages 111 - 118, XP027705948, ISSN: 1388-1981 * |
| LEE HA-REUM, SHIN SU-HYUN, KIM JOO HEON, SOHN KI-YOUNG, YOON SUN YOUNG, KIM JAE WHA: "1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG) Rapidly Resolves LPS-Induced Acute Lung Injury Through the Effective Control of Neutrophil Recruitment", FRONTIERS IN IMMUNOLOGY, vol. 10, 18 September 2019 (2019-09-18), XP055831101, DOI: 10.3389/fimmu.2019.02177 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3824886A4 (en) * | 2018-07-16 | 2022-04-20 | FNG Research Co., Ltd. | Novel compounds isolated from cervi parvum cornu, and pharmaceutical uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20210094996A (en) | 2021-07-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2013058484A2 (en) | Ingenane-type diterpene compound, and pharmaceutical composition for treating or preventing viral infectious diseases containing same | |
| WO2021206309A1 (en) | Pharmaceutical composition for prevention or treatment of diseases caused by sars-cov-2 | |
| WO2013089402A1 (en) | Composition comprising gypenoside extract of gynostemma pentaphyllum (thunb.) makino for treating or preventing type ιι diabetes, obesity, or hyperlipidemia | |
| WO2015160219A1 (en) | Pharmaceutical composition comprising pistacia weinmannifolia extract, fraction of same or compound separated from same for preventing or treating chronic obstructive pulmonary disease (copd) | |
| WO2011032491A1 (en) | Selfheal extraction containing rosmarinic acid, its preparation method and its use for preparation of medicine for precaution or treatment of cancer metastasis after surgery | |
| WO2021150076A1 (en) | Pharmaceutical composition or health functional food for preventing or treating chronic obstructive pulmonary diseases | |
| WO2021049864A1 (en) | Composition for ameliorating dry eye syndrome containing aralia elata extract | |
| WO2016167385A1 (en) | Composition-ii for preventing or treating alzheimer's disease, comprising lycopodiella cernua extract or compound isolated therefrom | |
| WO2021150077A1 (en) | Pharmaceutical composition or health functional food for prevention or treatment of non-alcoholic fatty liver disease | |
| WO2015026124A1 (en) | Composition containing monoacetyldiacylglycerol compound as active ingredient for preventing or treating asthma | |
| WO2013062247A2 (en) | Phorbol type diterpene compound, pharmaceutical composition for treatment or prevention of viral infectious diseases including same | |
| WO2015026123A1 (en) | Composition containing monoacetyldiacylglycerol compound as active ingredient for preventing or treating chronic obstructive pulmonary diseases | |
| WO2019231211A1 (en) | Composition for preventing or treating allergic diseases containing mixed extract of two or more among asiasarum root, platycodon root, and cinnamomi ramulus as active ingredients | |
| WO2013012117A1 (en) | Pharmaceutical compositions for preventing or treating inflammatory diseases, comprising phytosterol compound | |
| WO2022139529A1 (en) | Composition for preventing, improving or treating gastritis or peptic ulcer comprising extract of cinnamomum cassia, fraction of said extract, isolate of said fraction or compounds isolated therefrom | |
| WO2022045520A1 (en) | Composition for treating coronavirus-19 infection, comprising phenanthroindolizidine and phenanthroquinolizidine alkaloid derivative, optical isomer thereof, or pharmaceutically acceptable salt thereof as active ingredient | |
| WO2021091164A1 (en) | Phamaceutical composition for preventing or treating obesity or allergy comprising rose extract as active ingredient | |
| WO2012173392A2 (en) | Curcuminoid-based compound/stevioside-containing complex for the prevention and treatment of an influenza virus infection | |
| WO2020209476A1 (en) | Composition comprising clay mineral complex for prevention, alleviation, and treatment of inflammatory bowel disease, preparation method for composition, and method for alleviation and treatment for inflammatory bowel disease | |
| WO2010143919A2 (en) | Method and composition for preventing or treating il-6-mediated diseases or rhinovirus infections | |
| WO2019245245A1 (en) | Pharmaceutical composition for preventing and treating liver damage comprising turmeric extract | |
| WO2024117426A1 (en) | Composition for ameliorating or treating respiratory diseases caused by fine dust irritation containing adenophora stricta extract as active ingredient, and method for preparing same | |
| WO2021261870A1 (en) | Composition comprising 6'-hydroxy justcidin-b, for preventing, ameliorating or treating allergic diseases | |
| KR100643878B1 (en) | Pharmaceutical composition for inhibiting respiratory disease comprising old platycodon extracts as an effective components | |
| WO2023182540A1 (en) | Composition for preventing and ameliorating viral disease |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21744363 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 21744363 Country of ref document: EP Kind code of ref document: A1 |